High fructan cereal plants

ABSTRACT

The invention provides cereal plants having a high level of fructan useful for the production of a range of food, beverage, nutraceutical and pharmaceutical products. The invention provides methods of producing high-fructan products from plants modified to comprise a reduced level of an endogenous polypeptide with starch synthase activity, and products so produced. In some embodiments, plants are modified by introduction of an agent such as a nucleic acid molecule which down regulates endogenous starch synthase II gene expression.

This application is a divisional of U.S. Ser. No. 13/008,746, filed Jan.18, 2011, now allowed, which is a continuation-in-part of PCTInternational Application No. PCT/AU2009/000911, filed Jul. 16, 2009,which claims the benefit of U.S. Provisional Applications Nos.61/135,111, filed Jul. 17, 2008 and 61/135,361, filed Jul. 18, 2008, andclaims priority of Australian Patent Application No. 2009200577, filedFeb. 13, 2009, the contents of each of which are hereby incorporated byreference in their entirety into this application.

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“170808_79593-BZ_Substitute_Sequence_Listing_REL_txt” which is 114kilobytes in size, and which was created Aug. 8, 2017 in the IBM-PCTmachine format, having an operating system compatability withMS-Windows, which is contained in the text file filed Aug. 8, 2017 aspart of this application.

FIELD

The present specification describes cereal plants having a high level offructans useful for the production of a range of food, beverage,nutraceutical and pharmaceutical products.

BACKGROUND

Fructans are polymers of fructose which are synthesized from sucrose andused as storage or reserve carbohydrates by many plants. They consist offructosyl residues polymerized to sucrose, and therefore comprisefructosyl units in addition to one glucose unit. In view of thiscomposition, they are highly soluble in water. The linkages between thefructosyl-residues are either exclusively of the β(1-2) type forming alinear molecule (inulin) in which the fructosyl residues are attached tothe fructosyl residue of the sucrose starter, or of the β(2-6) type(levan), or both linkage types occur in branched fructans (graminans).Inulins are present in plants belonging to the Asterales (e.g. chicory)or the Liliaceae (e.g. onion). All fructans found in the dicotyledons,as well as some monocotyledons are of this type. The inulin in onion istermed neo-series inulin and has two linear β(1-2)-linked fructosylchains, one attached to the C1 of the fructosyl residue of the sucroseand one attached to the C6 of the glucosyl residue of the sucrose.Levans are typically found in monocotyledons such as the Poaceae (e.g.grasses) and in almost all bacterial fructans. Graminans which consistof β(2-6)-linked fructose units with β(1-2) branches and are thereforemore complex structures can also be present in cereals, and can be mixedwith levans.

The degree of polymerization (DP) and distribution of linkage types arecharacteristic of different plant species. Since a range of DP are oftenseen in any one species, fructans typically show a disperse molecularweight. In contrast to the high molecular weight of fructan (levan,1-5×10⁶ Da) elaborated as an extracellular polysaccharide by somebacteria, plant fructans are much smaller by 2-3 orders of magnitude.

Fructans, rather than starch, occur naturally as the primary reservecarbohydrate in about 10-15% of higher plants including chicory,artichoke, asparagus, dahlia and the onion family, primarily in theperennating organs. Fructans are mostly stored in taproots (e.g.chicory) or tubers (e.g. dahlia, Jerusalem artichoke) or bulbs (e.g.onion). In grasses and cereals, fructans are mainly stored in the stemsand leaf bases and used as a reserve carbohydrate for growth and seedproduction. Fructan also occurs as a temporary storage form in thevegetative tissues of forage grasses and cereals, but only at low levelsin cereal grain. Despite this, wheat products are the primary source offructan in the Western diet. Onions are the second largest source ofnaturally occurring fructans in the American diet, accounting for about25% of total consumption (Moshfegh et al., J Nutr. 129 (Suppl):1407S-11S, 1999).

Cereals such as wheat and barley accumulate, mainly in vegetativetissues, branched graminan-type fructans containing both β-(2,1) andβ-(2,6) fructosyl linkages. These mostly have a low DP, such as1-6-kestotetraose (bifurcose) which is the major fructan oligosaccharideaccumulating in crown tissues and leaves of cereals exposed to chilling.Fructans are naturally present in various cereal grains (White andSecor, Arch Biochem Biophys. 44: 244-5, 1953; Henry and Saini, CerealChem. 66: 362-365, 1989; Schnyder, New Phytol 123: 233-245, 1993). Wheatgrain has been reported to contain 0.6-2.6% (w/w) fructan.

Fructan is synthesized directly from sucrose as the sole precursor,without any known involvement of phosphorylated sugars or nucleotideco-factors, by the activity of specific fructosyltransferases (FTs).Synthesis generally occurs in vacuoles, outside of the plastid, andaccumulation of fructan occurs in vacuoles of both photosynthetic andstorage cells. Fructan synthesis in plants is initiated by asucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) usingsucrose both as fructosyl donor and acceptor to produce 1-kestose, theshortest β(1-2) linked fructan) and glucose. 1-SST is found in allfructan-producing plants. Longer chain inulins are formed by the actionof a second enzyme, fructan:fructan 6-fructosyltransferase (1-FFT, EC2.4.1.100) which adds fructosyl residues by β(1-2) linkages. 1-FFT canuse 1-kestose or fructans as fructose donors and therefore can transferfructosyl residues from one fructan chain to another. Synthesis of theneo-series fructans requires fructan:fructan 6G-fructosyltransferases(6G-FFT). In the case of cereals such as wheat and barley, the next stepof fructan synthesis is mediated by a sucrose:fructan6-fructosyltransferase (6-SFT, EC 2.4.1.10) which transfers a fructosylunit from a further sucrose molecule to fructan with a β(2-6) linkages,to extend the fructan polymer. Fructosyl transfer to 1-kestose, thesmallest branched fructan, forms the tetrasaccharide bifurcose. Itremains to be shown whether or not additional FTs are involved infructan synthesis of grasses or cereals, but the combined action of1-SST, 1-FFT, 6-FFT and 6G-FFT may be involved in graminan synthesis.

Many plant fructosyltransferases have been sequenced during the last fewyears, and the data clearly indicate a high homology to the vacuolar,acid invertases (β-fructosidases). These enzymes are all members of theglycoside hydrolase family 32 (GH32) and share three highly conservedregions characterized by the motifs (N/S)DPNG (also calledβ-fructosidase motif), RDP, and EC. The aspartate of the (N/S)DPNG motifprovides a nucleophile in the catalysis, the glutamate of the EC-motifas a proton donor, and the aspartate of the RDP motif as transitionstate stabilizer in the transfructosylation reaction.

Fructans are catabolised by fructan exohydrolases (FEH; EC 3.2.1.80)which are specialized for fructans, and invertases such as acidinvertase (EC 3.2.1.26) which hydrolyse sucrose. Genes encoding fructanexohydrolase have been isolated from wheat (Van den Ende et al., PlantPhysiol. 131(2): 621-631, 2003). Other enzymes such as sucrose phosphatesynthase (SPS; EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) areassociated with fructan remobilization from the stems.

Fructans are non-starch carbohydrates with potentially beneficialeffects as a food ingredient on human health (Tungland and Meyer,Comprehensive Reviews in Food Science and Food Safety, 2: 73-77, 2002;Ritsema and Smeekens, Curr. Opin. Plant Biol. 6: 223-230, 2003). Thehuman digestive enzymes α-glucosidase, maltase, isomaltase and sucraseare not able to hydrolyse fructans because of the β-configuration of thefructan linkages. Furthermore, humans and other mammals lack the fructanexohydrolase enzymes that break down fructans and therefore dietaryfructans avoid digestion in the small intestine and reach the largeintestine intact. However, bacteria there are able to ferment fructansand thereby utilize them as, for example, an energy or carbon source forgrowth and production of short-chain fatty acids (SCFA). Dietaryfructans therefore are able to stimulate the growth of beneficialbacteria such as bifidobacteria in the colon, which aids in preventionof bowel disorders such as constipation and infection by pathogenic gutbacteria. Dietary fructan also enhances nutrient absorption from diets,particularly calcium and iron, possibly via production of SCFA which inturn reduce luminal pH and modify calcium speciation and hencesolubility, or exert a direct effect on the mucosal transport pathway,thereby improving the mineralization of bone and reducing the risk ofiron deficiency anaemia. In addition, a high-fructan diet can improvethe health of patients with diabetes and reduce the risk of coloniccancers by suppressing aberrant crypt foci which are precursors of coloncancer (Kaur and Gupta, J. Biosci. 27: 703-714, 2002).

Attempts have been made to enhance fructan production in transgenicplants by introduction and expression of genes encoding 1-SST and 1-FFT.Generally, fructan accumulation levels were less than 2% (w/w) forplants transformed with bacterial genes and less than 1% (w/w) usingplant genes. In some exceptions, concentrations of 6-16% on a freshweight basis were achieved and compare favourably with naturallyoccurring maximal starch and fructan content in leaves and tubers(Sevenier et al., Nature Biotechnol. 16: 843-846, 1998; Hellwege et al.,Proc. Natl. Acad. Sci. U.S.A. 97: 8699-8704, 2000). Transformantsexpressing bacterial fructan synthesis genes sometimes exhibitedaberrant phenotypes such as stunting, leaf bleaching, necrosis, reducedtuber number and mass, tuber cortex discoloration, reduction in starchaccumulation, and chloroplast agglutination.

There is therefore a need for efficient production of fructan from plantsources at low cost.

SUMMARY

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

As used herein the singular forms “a”, “an” and “the” include pluralaspects unless the context clearly dictates otherwise. Thus, forexample, reference to “a mutation” includes a single mutation, as wellas two or more mutations; reference to “an agent” includes one agent, aswell as two or more agents; and so forth.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of sequence identifiers is provided in Table 7. Asequence listing is provided after the claims.

Genes and other genetic material (e.g. mRNA, nucleic acid constructsetc) are represented herein in italics while their proteinaceousexpression products are represented in non-italicised form. Thus, forexample starch synthase II (SSII) polypeptide is the expression productof SSII nucleic acid sequences.

Representative examples of the nucleic acid and amino acid sequences ofSSII molecules are provided in the sequence listing further described inTable 7. The terms SSII or SSII encompass all functional homologs in anyplant species including cereal plants and including SSII molecules suchas SSIIa, SSIIb, SSIIa-2, SSIIa-B, SSIIa-D and SSII-2 etc. In aparticular embodiment, the SSII is SSIIa.

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

Each embodiments described herein is to be applied mutatis mutandis toeach any every embodiment unless specifically stated otherwise.

Accordingly in one embodiment, the present specification describes amethod of producing a high-fructan product, wherein the methodcomprises: (i) obtaining or producing a cereal plant or grain or flourtherefrom wherein the cereal grain or flour comprises at least 3%,preferably at least 4%, fructan as a percentage of the cereal grain orflour weight; and (ii) processing the plant, grain or flour to producethe product.

In particular embodiments, the grain is characterized by a combinationof two parameters: the percent fructan in the grain by weight, and thestarch content of the grain by weight. For the first parameter, thepercentage fructan of the cereal grain by weight is at least 3%,preferably at least 4%, at least 5%, at least 6%, at least 7%, at least8%, at least 9% or at least 10% as shown herein For the secondparameter, the starch content of the grain by weight, is at least 30%,preferably at least 35%, at least 36%, at least 37%, at least 38%, atleast 39% or at least 40% as a percentage of the total grain weight. Theinvention includes each and every specific combination of these twoparameters with respect to the grain, products obtained therefrom,methods of obtaining and using the grain, and uses of the grain andproducts therefrom. Reference herein to “high fructan” is used merely toindicate that the product is produced from the herein disclosed modifiedgrain having an elevated level of fructan compared to an unmodified orcontrol form of the grain.

In another embodiment, the method comprises: (i) obtaining or producinga cereal plant or grain or flour therefrom wherein wherein the cerealplant or grain is modified to comprise a reduced level of an endogenouspolypeptide with starch synthase II (SSII) activity relative to anunmodified control; and (ii) processing the plant, grain or flour toproduce the product. Various forms of this aspect of the invention aredescribed in the Examples. Methods for reducing the level of anendogenous polypeptide with starch synthase II (SSII) activity in cerealplants relative to an unmodified or control plant are known in the artas described herein and in the common general knowledge. In particularembodiments, the grain is characterized by the same combination of twoparameters as described in paragraph 0021.

In some embodiments, the methods further comprise: (iii) assessing thelevel or type of fructan in the cereal plant or grain or flourtherefrom, or the product therefrom.

In several embodiments, the cereal grain is wholegrain which may becracked, ground, polished, milled, kibbled, rolled or pearled grain. Insome embodiments, the present invention extends to monocotyledonouscereal plants selected from the group consisting of barley, wheat, rice,maize, rye, oat and sorghum. In further embodiments, the plant istetraploid wheat, maize, rye, rice, oat or sorghum, or hexaploid wheator barley.

In a particular embodiment, the plant is a barley shrunken grain mutantdesignated M292 or M342 described in International Publication No. WO02/37955.

As illustrated in the Examples, the fructan of the present invention insome embodiments comprises a degree of polymerization from about 3 toabout 12.

In one application of the present invention, the product is a food orbeverage product or a pharmaceutical composition. In a particularembodiment, the product is isolated fructan. non-limiting examples offood or beverage products include, grain, flour, breakfast cereal,biscuit, muffin, muesli bar, noodle, corn, a sweetening agent, a lowcalorie additive, a bulking agent, a dietary fibre, a texturizing agent,a preservative, a probiotic agent or the like or any combination ofthese.

In some further embodiments, the cereal plant or grain of the presentinvention is modified to comprise a reduced level of an endogenouspolypeptide with starch synthase II (SSII) activity relative to anunmodified control. As known to those of skill in the art a wide rangeof methods are available for reducing the level of an endogenouspolypeptide in a plant. In some embodiments, the plant comprises amutation in an endogenous gene encoding a polypeptide with SSII activitywherein the mutation reduces the expression of the gene encoding SSII inthe plant or leads to the expression of SSII with reduced level oractivity. In other embodiments, the level of SSII activity is reduced byintroducing into said plant a nucleic acid molecule which down-regulatesthe expression of a gene encoding SSII in the plant. In someembodiments, the nucleic acid molecule comprises a gene-silencingchimeric gene, an antisense, ribozyme, co-expression dsRNA molecule, orother exogenous nucleic acid molecule that down-regulates endogenousSSII expression. In preferred embodiments, the grain is characterized bythe same combination of two parameters as described in paragraph 0021.

In another aspect the present invention provides a method of producing acereal plant or grain therefrom which has increased levels of fructancompared to a control plant, wherein the method comprises: (i)introducing into said plant an agent which down-regulates the level oractivity of endogenous starch synthase II (SSII) in the plant relativeto a control plant, or a mutation in an endogenous gene encoding SSII inthe plant. As described further herein in some embodiments, the agentcomprises a nucleic acid molecule which down-regulates endogenous SSIIgene expression. Illustrative nucleic acid molecules include agene-silencing chimeric gene, an antisense, ribozyme, co-expressiondsRNA molecule, or other exogenous nucleic acid molecule thatdown-regulates endogenous SSII expression.

In a further embodiment of this aspect of the invention the methodcomprises assessing the level, activity or type of fructan in the plantor grain therefrom. In some embodiments, the increased level of fructanis at least twice, preferably at least 3×, at least 4×, at least 5×, atleast 6×, at least 7×, at least 8×, at least 9× or at least 10×, that ofa control plant or the plant prior to the introduction of the agent ormutation.

In another aspect, the present specification provides an isolated orgenetically modified cereal plant or grain or flour therefrom whereinthe grain or flour comprises at least 3%, preferably at least 4% fructanas a percentage of the cereal grain or flour weight. Preferably, theplant or grain or flour is used, or is for use, in the production of aproduct to increase the level of fructan or non-starch carbohydrate insaid product. In some embodiments, the percentage fructan of the cerealgrain or flour by weight is at least 5%, at least 6%, at least 7%, atleast 8%, at least 9% or at least 10% as shown herein. In preferredembodiments, the grain is characterized by the same combination of twoparameters as described in paragraph 0021. As is readily apparent, theinvention includes the flour, fructan and food products produced fromeach of these preferred embodiments of grain.

Accordingly, the present invention contemplates, cereal grain, flour orfructan produced from the plant or grain as described herein. In someembodiments, the cereal grain or flour comprises a starch content of atleast 30%, preferably at least 35%, at least 36%, at least 37%, at least38%, at least 39% or at least 40% as a percentage of the total grainweight.

In some embodiments, the cereal plant is not barley or hexaploid wheat.

In particular embodiments, the cereal grain or flour as described hereincomprising a reduced level or activity of a polypeptide having SSIIactivity.

In some embodiments, the present invention provides fructan, grain orflour produced from the plant or grain or flour as described herein. Theinventors contemplate, for example, the use of fructan isolated from thesubject plant, grain or flour in a food as a sweetening agent, a lowcalorie additive, a bulking agent, a dietary fibre, a texturizing agent,a preservative, a probiotic agent or the like or any combination ofthese. In some embodiments, the inventors contemplate the use of a grainor flour or frunctan isolated from a plant, grain or flour as describedherein in the production of a food product to increase the level offrunctan in said food product. In preferred embodiments, the grain ischaracterized by the same combination of two parameters as described inparagraph 0021.

In some embodiments, the food product comprises a food ingredient at alevel of at least 10% on a dry weight basis, wherein the food ingredientis a cereal grain comprising at least 3%, preferably at least 4%,fructan on a weight basis or wholemeal or flour obtained therefromwherein the wholemeal or flour comprises at least 3%, preferably atleast 4% fructan on a weight basis. In preferred embodiments, the grainis characterized by the same combination of two parameters as describedin paragraph 0021.

In yet another aspect, the present invention provides a method ofidentifying a variety of cereal grain which has increased levels offructan comprising: (i) obtaining cereal grain which is altered instarch via synthesis or catabolism; (ii) determining the amount offructan in the grain, (iii) comparing the level of fructan to that inwild-type grain which is not altered in starch via synthesis orcatabolism, and (iv) if the fructan level is increased in the alteredgrain, selecting the grain. In some embodiments, the method furthercomprises mutagenesis or plant cell transformation prior to step (i).

In another embodiments, a method is provided for determining the amountof fructan in cereal grain, comprising the steps of (i) obtaining graincomprising at least 3%, preferably at least 4% fructan on a weightbasis, processing the grain so as to extract the fructan, and measuringthe amount of extracted fructan so as to determine the amount of fructanin the grain.

In a further embodiment, the present invention contemplates a method forpreparing a food or beverage, comprising mixing a high-fructan productobtained by the herein disclosed methods with another food or beverageingredient.

In another embodiment, the present invention provides a method forproviding fructan to improve one or more indicators of health in asubject in need thereof, wherein the method comprises administering, tothe subject, a composition comprising cereal grain or flour therefromcomprising at least 3%, preferably at least 4%, fructan on a weightbasis or fructan obtained therefrom. In some embodiments, the grain,flour or fructan is in the form of a food product, a beverage or apharmaceutical composition. In other embodiments, the one or moreindicators of health is an increased number of beneficial intestinalbacteria, reduced number of aberrant crypt foci, increased mineralabsorption, reduced level of insulin, reduced glycaemic index, reducedglycaemic load, reduced blood glucose, reduced blood pressure, reducedbody weight, reduced blood cholesterol level, increased HDL cholesterollevel, increased bone density, increased calcium levels, more frequentbowel movement, or improved blood serum cardiovascular profile. Inpreferred embodiments, the grain is characterized by the samecombination of two parameters as described in paragraph 0021.

The present invention provides a method for ameliorating one or moresymptoms of a condition associated with low levels of dietary fructan ina subject in need thereof, said method comprising administering orallyto the subject grain comprising at least 3%, preferably at least 4%,fructan as a percentage of the cereal grain weight or a processedproduct comprising the fructan obtained therefrom for a time and underconditions sufficient to ameliorate one or more symptoms. The condition,in some embodiments, is selected from the group consisting of diabetes,obesity, heart disease, hypertension, constipation, osteoporesis andcancer. The method may comprise the step of determining that the subjectmay benefit from increased intake of dietary fructan. In preferredembodiments, the grain is characterized by the same combination of twoparameters as described in paragraph 0021.

Any subject who could benefit from the present methods or compositionsis encompassed. The term “subject” includes, without limitation, humansand non-human primates, livestock animals, companion animals, laboratorytest animals, captive wild animals, reptiles and amphibians, fish, birdsand any other organism. A subject, regardless of whether it is a humanor non-human organism may be referred to as a patient, individual,subject, animal, host or recipient. In a particular embodiment thesubject is a human.

The above summary is not and should not be seen in any way as anexhaustive recitation of all embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation showing the soluble carbohydrateprofile of mutant ‘M292’ and wild type ‘Himalaya’ mature barley grains.HPAE chromatographic elution profile of grain extracts indicatingincreased levels of hexose, sucrose, maltose and fructo-oligosaccharidesin the mutant M292 compared to wild type Himalaya grain. Glc, glucose;Fru, fructose; Suc, sucrose; Mal, maltose; 1-K, 1-kestose; numeralsindicate tentative degree of polymerization (DP) offructo-oligosaccharides.

FIG. 2 is a graphical representation of data showing the fermentationproperties of modified cereal products. Extract of barley grain mutant292 and standards (inulin, lactulose, glucose) were fermented incarbon-limited fermentation media. Short chain fatty acid (SCFA)production was measured. This indicated that the lower doses of mutant292 produced comparable fermentation to that of inulin (see total SCFA1^(st) & 2^(nd) column (1% 292 and inulin 1%) and 5^(th) & 6^(th) column(2% 292 and Inulin 2%).

FIG. 3 is a graphical representation of data showing in vivo effect inrats of a diet including barley grain 292 compared to correspondingoligofructose standards and controls. The total amount of short chainfatty acids and acidification of caecal digesta was greater fortreatments than controls and barley grain 292 produced comparativeresults to those of oligofructose standards. Items in key for SCFA typesrunning top to bottom correspond to columns of bar graph for eachtreatment running from left to right.

DETAILED DESCRIPTION

The present specification was based at least in part upon the discoverythat cereal barley plants having reduced levels of synthesis ofamylopectin through down regulation of starch synthase II geneexpression also exhibit low levels of amylopectin, a relatively highproportion of amylose in the total starch of the grain, enhanced levelsof sugars and surprisingly high levels of non-starch polysaccharide(NSP) and particularly fructan on a weight basis (see Table 1 and Table3). Transcriptional profiles of plants comprising a loss of functionmutation in SSII or SSII and exhibiting high fructan levels are shown inTable 2.

Accordingly, in one embodiment, the specification provides a method ofproducing a high-fructan product, wherein the method comprises: (i)obtaining or producing a cereal plant or grain or flour therefromwherein the cereal grain or flour comprises at least 3%, preferably atleast 4%, fructan as a percentage of the cereal grain or flour weight;(ii) processing the plant, grain or flour to produce the product, andoptionally (iii) assessing the level or type of fructan in the cerealplant or grain or flour therefrom, or the product therefrom. It isbelieved that the presence of 3% or 4% fructan in the grain of cerealdistinguishes the present invention from the prior art. However,illustrative levels of at least about 10% fructan are described andaccordingly, the percentage fructan of the cereal grain or flour byweight in some embodiments, is at least 5%, at least 6%, at least 7%, atleast 8%, at least 9% or at least 10%. If the product is isolatedfructan, the product may comprise at least 50%, or at least 60% or atleast 70% or preferably at least 80% fructan by weight.

In another embodiment, the method comprises: (i) obtaining or producinga cereal plant or grain or flour therefrom wherein wherein the cerealplant or grain is modified to comprise a reduced level of an endogenouspolypeptide with starch synthase II (SSII) activity relative to anunmodified control; (ii) processing the plant, grain or flour to producethe product, and optionally (iii) assessing the level or type of fructanin the cereal plant or grain or flour therefrom, or the producttherefrom.

It is unexpected that reduction in starch synthase activity whichreduces the formation of amylopectin, also increases fructan productionin the plant. Starch serves as the primary carbohydrate component in thediet of humans, in particular from cereals. Starch is the major storagecarbohydrate in cereals, making up approximately 45-65% of the weight ofthe mature grain. However, cereal grains also contain non-starchpolysaccharides such as β-glucans or low levels of fructans. Inwild-type wheat grain, the level of fructan is only 0.6%-2.6% by weight.

Starch is composed only of glucosidic residues and is found as two typesof molecules, amylose and amylopectin, which can be distinguished on thebasis of molecular size or other properties. Amylose molecules areessentially linear polymers composed of α-1,4 linked glucosidic units,while amylopectin is a highly branched molecule with α-1,6 glucosidicbonds linking many linear chains of α-1,4 linked glucosidic units.Amylopectin is made of large molecules ranging in size between severaltens of thousands to hundreds of thousands of glucose units with around5 percent α-1,6 branches. Amylose on the other hand is composed ofmolecules ranging in size between several hundreds to several thousandglucosidic residues with less than one percent branches (for review seeBuléon et al., International Journal of Biological Macromolecules, 23:85-112, 1998). Wild-type cereal starches typically contain 20-30%amylose while the remainder is amylopectin.

The synthesis of starch in the endosperm of higher plants is carried outby a suite of enzymes that catalyse four key steps. Firstly, ADP-glucosepyrophosphorylase activates the monomer precursor of starch through thesynthesis of ADP-glucose from G-1-P and ATP. Secondly, the activatedglucosyl donor, ADP-glucose, is transferred to the non-reducing end of apre-existing α1-4 linkage by starch synthases. Thirdly, starch branchingenzymes introduce branch points through the cleavage of a region ofα-1,4 linked glucan followed by transfer of the cleaved chain to anacceptor chain, forming a new α-1,6 linkage. Starch branching enzymesare the only enzymes that can introduce the α-1,6 linkages intoα-polyglucans and therefore play an essential role in the formation ofamylopectin. Finally, starch debranching enzymes remove some of thebranch linkages although the mechanism through which they act isunresolved.

While it is clear that at least these four activities are required fornormal starch granule synthesis in higher plants, multiple isoforms ofeach of the four activities are found in the endosperm of higher plantsand specific roles have been proposed for individual isoforms on thebasis of mutational analysis or through the modification of geneexpression levels using transgenic approaches. In the cereal endosperm,four classes of starch synthase are found in the cereal endosperm, anisoform exclusively localised within the starch granule, granule-boundstarch synthase (GBSS) which is essential for amylose synthesis, twoforms that are partitioned between the granule and the soluble fraction(SSI, Li et al., Plant Physiology, 120: 1147-1155, 1999a, SSII, Li etal., Theoretical and Applied Genetics, 98: 1208-1216, 1999b) and afourth form that is entirely located in the soluble fraction, SSIII (Caoet al., Archives of Biochemistry and Biophysics, 373: 135-146, 2000; Liet al., 1999b (supra); Li et al., Plant Physiology, 123: 613-624, 2000).Mutations in SSII and SSIII have been shown to alter amylopectinstructure (Gao et al., Plant Cell, 10: 399-412, 1998; Craig et al.,Plant Cell 10: 413-426, 1998). No mutations defining a role for SSIactivity have been described.

Three forms of branching enzyme are expressed in the cereal endosperm,branching enzyme I (SBEI), branching enzyme IIa (SBEIIa) and branchingenzyme IIb (SBEIIb) (Hedman and Boyer, Biochemical Genetics, 20:483-492, 1982; Boyer and Preiss, Carbohydrate Research, 61: 321-334,1978; Mizuno et al., Journal of Biochemistry, 112: 643-651, 1992; Sun etal., The New Phytologist, 137: 215-215, 1997). Alignment of SBEsequences has revealed a high degree of sequence similarity at both thenucleotide and amino acid levels and allows the grouping into the SBEI,SBEIIa and SBEIIb classes.

Two types of debranching enzymes are present in higher plants and aredefined on the basis of their substrate specificities, isoamylase typedebranching enzymes, and pullulanase type debranching enzymes (Myers etal., Plant Physiology, 122: 989-997, 2000). Sugary-1 mutations in maizeand rice are associated with deficiency of both debranching enzymes(James et al., Plant Cell, 7: 417-429, 1995; Kubo et al., PlantPhysiology, 121: 399-409, 1999) however the causal mutation maps to thesame location as the isoamylase-type debranching enzyme gene.

A mutant form of barley, designated M292 or M342, has been shown to havean elevated amylose starch phenotype and a reduced amylopectin starchphenotype. This phenotype has suspected benefits for human health(Morell et al., Plant J. 34: 173-185, 2003; Topping et al.,Starch/Stärke 55: 539-545, 2003; Bird et al., J. Nutr. 134: 831-835,2004a; Bird et al. Br. J. Nutr. 92: 607-615, 2004b). It is caused by amutation in the starch synthase IIa gene (SSIIa) located on chromosome7H of barley, as described in international patent applicationPCT/AU01/01452 (Publication No. WO 02/37955) the disclosure of which isincorporated herein by reference.

The barley sex6 mutation resulted from the presence of a stop codonwithin the starch synthase IIa (SSIIa) gene. The stop codon lead topremature termination of translation of the transcript. The SSIIaprotein was not detectable in the endosperm of this mutant (Morell etal. 2003 (supra)). The loss of SSIIa activity lead to an 80% decrease inamylopectin synthesis, and the remaining amylopectin polymers in generalhave altered chain length distribution, and consequently an alteredamylose:amylopectin ratio so that the starch of the grain containedabout 70% amylose.

SSII mutants of wheat have also been produced (Yamamori et al., Theor.Appl. Genet. 101: 21-29, 2000) although the amylose level was not ashigh as in the barley mutant, reaching about 38% in the wheat. Incontrast, down-regulation of the genes encoding SBEIIa in wheat resultedin a high amylose phenotype, with about 80% amylose in the starch of thegrain (Regina et al., Proc. Natl. Acad. Sci. U.S.A. 103: 3546-3551,2006).

In some embodiments, the present invention provides for improvements incereal plant utility by increasing the level of fructan in grain. Themodification may be limited to grain or alternatively, the modificationmay be thoughout the plant in various of its tissues and parts. As usedherein, “modifying” or “modified” means a change in the plant or grain,which may be an increase or decrease in amount, activity, rate ofproduction, rate of inactivation, rate of breakdown, delay of onset,earlier onset, addition or removal of material, mutation, or anycombination of these, so long as there is a reduced level or activity ofstarch synthase II. The terms include either an increase or decrease inthe functional level of a gene or protein of interest. “Functionallevel” should be understood to refer to the level of active protein. Thefunctional level is a combination of the actual level of protein presentin the host cell and the specific activity of the protein. Accordingly,the functional level may e.g. be modified by increasing or decreasingthe actual protein concentration in the host cell, which may readily beachieved by altering expression of a gene encoding the protein. Thefunctional level may also be modified by modulating the specificactivity of the protein. Such increase or decrease of the specificactivity may be achieved by expressing a variant protein with higher orlower specific activity or by replacing the endogenous gene encoding therelevant protein with an allele encoding such a variant. Increase ordecrease of the specific activity may also be achieved by expression ofan effector molecule. In certain embodiments, the expression level of anappropriate coding sequence or activity or amount of an enzyme is chosensuch that it is at least about 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 80% or even at least about100%, at least 200%, at least 500%, or at least 1000% higher, or atleast about 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 97%, at least 98% or at least 99%lower than a reference expression level, or reduced to an undetectablelevel.

Another way of distinguishing the required reduction in SSII level oractivity is by quantifying the increase level or the increase in variousforms of fuctan in a modified plant or grain therefrom. As used herein,the terms “modifying”, “altering”, “increasing”, “increased”,“reducing”, “reduced”, “inhibited”, “mutant” or the like are consideredrelative terms, i.e. in comparison with the wild-type or unaltered orcontrol state. In some embodiments, a wild-type plant is an appropriate“control plant” however in many situations the control plant must bedetermined by the skilled addressee using their ordinary skill in theart. The “level of a protein” refers to the amount of a particularprotein, for example SSII, which may be measured by any means known inthe art such as, for example, Western blot analysis or otherimmunological means. The “level of an enzyme activity” refers to theamount of a particular enzyme measured in an enzyme assay.

It would be appreciated that the level of activity of an enzyme might bealtered in a mutant if a more or less active protein is produced, butnot the expression level (amount) of the protein itself. Conversely, theamount of protein might be altered but the activity (per unit protein)remain the same. Reductions in both amount and activity are alsopossible such as, for example, when the expression of a gene encodingthe enzyme is reduced transcriptionally or post-transcriptionally. Incertain embodiments, the reduction in the level of protein or activityof SSII is by at least 40% or by at least 60% compared to the level ofprotein or activity in the grain of unmodified cereal, for example wheator barley, or by at least 75%, at least 90% or at least 95%. Thereduction in the level of the protein or enzyme activity or geneexpression may occur at any stage in the development of the leaf, seedor grain, particularly during the daytime when photosynthesis isoccurring, or during the grain filling stage while starch is beingsynthesized in the developing endosperm, or at all stages of graindevelopment through to maturity. The term “wild-type” as used herein hasits normal meaning in the field of genetics and includes plant,preferably cereal, cultivars or genotypes which are not modified astaught herein. Some preferred “wild-type” cereal plant varieties aredescribed herein.

The modified phenotype may be achieved by partial or full inhibition ofthe expression of an SSII gene. Techniques well known in the art such asSDS-PAGE and immunoblotting are carried out on hydrolysed andunhydrolysed grains and fractions thereof to identify the plants orgrain where modifications have occurred to starch forming enzymes,carbohydrate related genes, defense related genes, stress proteinrelated genes or genes identified as differentially expressed in thesubject modified plants or grain thereform (such as those listed inTable 2). These methods include analysis of plants by methods describedherein or further by methods such as such as microarray analysis,electrophoresis, chromatography (including paper chromatography, thinlayer chromatography, gas chromatography, gas-liquid chromatography andhigh-performance liquid chromatography) techniques. Separated componentsare typically identified by comparison of separation profiles withstandards of known identity, or by analytical techniques such as massspectrometry and nuclear magnetic resonance spectroscopy. For example,reference may be made to Example 9, Robinson, The Organic Constituentsof Higher Plants, Cordus Press, North Amherst, USA, 1980; Adams et al.,Anal. Biochem., 266: 77-84, 1999; Veronese et al., Enz. Microbial Tech.,24: 263-269, 1999; Hendrix et al., J. Insect Physiol., 47: 423-432,2001; Thompson et al., Carbohydrate Res., 331: 149-161, 2001; andreferences cited therein. Carbohydrates can be assayed using standardprotocols known to persons skilled in the art.

Alteration in SSII activities may be achieved by the introduction of oneor more genetic variations into the cereal plant. That is, the geneticvariations lead, directly or indirectly, to the alteration in enzymeactivity in the plant part during growth or development and consequentlyto the enzyme and fructan modifications described herein. The geneticvariation may be a heterologous polynucleotide which is introduced intothe plant or a progenitor cell, for example by transformation ormutagenesis. The genetic variation may subsequently be introduced intodifferent genetic backgrounds by crossing, as known in the art of plantbreeding. In some embodiments, the level or functional activity of SSIIis down regulated to a level less than about 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20% orless than 15%, and suitably less than about 10%, less than 9%, less than8%, less than 7%, less than 6%, less than 5%, less than 4%, less than3%, less than 2% or less than 1% relative to a corresponding controlplant to achieve elevated levels of fructan. In a preferred embodiment,elevated levels are at least twice that of controls. Preferably, in thisembodiment, this reduction results in a substantial enhancement ofnon-starch polysaccharide such as fructan levels which is generally atleast about 50% or 55% and more especially at least about 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% or greater increase in fructan level relative to acorresponding control plant grown under the same environmentalconditions. The amount of reduced SSII level or activity required maydepend upon other factors such as the plant species or strainenvironmental factors. However, it is considered that any optimisation,which may be required in such an event is achievable using routinemethods including those described herein.

Reduced SSII levels may be accomplished in tissues throughout the plant,for example using a constitutive promoter to drive expression of aheterologous polynucleotide that down regulates SSII. Alternatively, itmay be accomplished in source tissues (leaves), in transport tissues orin sink tissues (endosperm) using a tissue-specific or developmentallyregulated promoter. “Sink cell” and “sink tissue” as used herein, referto cells, tissues or organs which comprise a net inflow of organiccarbon that has entered the cells in a form other than fixation ofcarbon dioxide ie. as sugars or other carbohydrates. In plants, sinktissues include all non-photosynthetic tissues, as well asphotosynthetic tissues with a net inflow of organic carbon fixed byother photosynthetic cells or otherwise obtained from the surroundingmedium or environment by means other than direct fixation of carbondioxide.

In certain embodiments, the level fructan in grain is increased at leastabout 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80% or at least 90%, or even at leastabout 100%, at least 200%, at least 300%, at least 400%, at least 500%,at least 600%, at least 700%, at least 800%, at least 900% or at least1000% higher relative to controls.

Genes

In some embodiments, the present invention involves modification of geneactivity and the construction and use of chimeric genes. As used herein,the term “gene” includes any deoxyribonucleotide sequence which includesa protein coding region or which is transcribed in a cell but nottranslated, as well as associated non-coding and regulatory regions.Such associated regions are typically located adjacent to the codingregion or the transcribed region on both the 5′ and 3′ ends for adistance of about 2 kb on either side. In this regard, the gene mayinclude control signals such as promoters, enhancers, termination and/orpolyadenylation signals that are naturally associated with a given gene,or heterologous control signals in which case the gene is referred to asa “chimeric gene”. The sequences which are located 5′ of the codingregion and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene.

The “starch synthase II gene” “SSII” or the like as used herein refersto a nucleotide sequence encoding starch synthase II (SSII) in cerealssuch as barley or wheat, which can readily be distinguished from otherstarch synthases or other proteins by those skilled in the art. WheatSSII genes include the naturally occurring variants existing in wheat,including those encoded by the A, B and D genomes of breadwheat, as wellas non-naturally occurring variants which may be produced by thoseskilled in the art of gene modification. In a preferred embodiment, abarley SSII gene refers to a nucleic acid molecule, which may be presentin or isolated from barley or derived therefrom, comprising nucleotideshaving a sequence having at least 80% identity to the cDNA sequenceshown in SEQ ID NO: 1. In another preferred embodiment, a wheat SSIIgene refers to a nucleic acid molecule, which may be present in orisolated from wheat or derived therefrom, comprising nucleotides havinga sequence having at least 80% identity to the cDNA shown in SEQ ID NO:3, 5, 7, 9 or 18. In a preferred embodiment, the SSII gene is an SSIIagene, or the SSII protein is an SSIIa protein, each of which may beapplied to any or all of the aspects of the invention disclosed herein.

A genomic form or clone of a gene containing the transcribed region maybe interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” An “intron” as usedherein is a segment of a gene which is transcribed as part of a primaryRNA transcript but is not present in the mature mRNA molecule. Intronsare removed or “spliced out” from the nuclear or primary transcript;introns therefore are absent in the messenger RNA (mRNA). Introns maycontain regulatory elements such as enhancers. “Exons” as used hereinrefer to the DNA regions corresponding to the RNA sequences which arepresent in the mature mRNA or the mature RNA molecule in cases where theRNA molecule is not translated. An mRNA functions during translation tospecify the sequence or order of amino acids in a nascent polypeptide.The term “gene” includes a synthetic or fusion molecule encoding all orpart of the proteins of the invention described herein and acomplementary nucleotide sequence to any one of the above. A gene may beintroduced into an appropriate vector for extrachromosomal maintenancein a cell or for integration into the host genome.

As used herein, a “chimeric gene” refers to any gene that is not anative gene in its native location. Typically a chimeric gene comprisesregulatory and transcribed or protein coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. The term “endogenous” is used herein to refer to a substancethat is normally present or produced in an unmodified plant at the samedevelopmental stage as the plant under investigation. An “endogenousgene” refers to a native gene in its natural location in the genome ofan organism. As used herein, “recombinant nucleic acid molecule” refersto a nucleic acid molecule which has been constructed or modified byrecombinant DNA technology. The terms “foreign polynucleotide” or“exogenous polynucleotide” or “heterologous polynucleotide” and the likerefer to any nucleic acid which is introduced into the genome of a cellby experimental manipulations. These include gene sequences found inthat cell so long as the introduced gene contains some modification(e.g. a mutation, the presence of a selectable marker gene, etc.)relative to the naturally-occurring gene. Foreign or exogenous genes maybe genes that are inserted into a non-native organism, native genesintroduced into a new location within the native host, or chimericgenes. A “transgene” is a gene that has been introduced into the genomeby a transformation procedure. The term “genetically modified” includesintroducing genes into cells by transformation or transduction, mutatinggenes in cells and altering or modulating the regulation of a gene in acell or organisms to which these acts have been done or their progeny.

Polynucleotides

The present invention including the description, tables and sequencelisting, refers to various polynucleotides. As used herein, a“polynucleotide” or “nucleic acid” or “nucleic acid molecule” means apolymer of nucleotides, which may be DNA or RNA or a combinationthereof, and includes mRNA, cRNA, cDNA, tRNA, siRNA, shRNA and hpRNA. Itmay be DNA or RNA of cellular, genomic or synthetic origin, for examplemade on an automated synthesizer, and may be combined with carbohydrate,lipids, protein or other materials, labelled with fluorescent or othergroups, or attached to a solid support to perform a particular activitydefined herein, or comprise one or more modified nucleotides not foundin nature, well known to those skilled in the art. The polymer may besingle-stranded, essentially double-stranded or partly double-stranded.An example of a partly-double stranded RNA molecule is a hairpin RNA(hpRNA), short hairpin RNA (shRNA) or self-complementary RNA whichinclude a double stranded stem formed by basepairing between anucleotide sequence and its complement and a loop sequence whichcovalently joins the nucleotide sequence and its complement. Basepairingas used herein refers to standard basepairing between nucleotides,including G:U basepairs. “Complementary” means two polynucleotides arecapable of basepairing (hybridizing) along part of their lengths, oralong the full length of one or both. A “hybridized polynucleotide”means the polynucleotide is actually basepaired to its complement. Theterm “polynucleotide” is used interchangeably herein with the term“nucleic acid”.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Asused herein, an “isolated polynucleotide” or “isolated nucleic acidmolecule” means a polynucleotide which is at least partially separatedfrom, preferably substantially or essentially free of, thepolynucleotide sequences of the same type with which it is associated orlinked in its native state. For example, an “isolated polynucleotide”includes a polynucleotide which has been purified or separated from thesequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment. Preferably, the isolated polynucleotide isalso at least 90% free from other components such as proteins,carbohydrates, lipids etc. The term “recombinant polynucleotide” as usedherein refers to a polynucleotide formed in vitro by the manipulation ofnucleic acid into a form not normally found in nature. For example, therecombinant polynucleotide may be in the form of an expression vector.Generally, such expression vectors include transcriptional andtranslational regulatory nucleic acid operably connected to thenucleotide sequence.

The present invention refers to use of oligonucleotides. As used herein,“oligonucleotides” are polynucleotides up to 50 nucleotides in length.They can be RNA, DNA, or combinations or derivatives of either.Oligonucleotides are typically relatively short single strandedmolecules of 10 to 30 nucleotides, commonly 15-25 nucleotides in length.When used as a probe or as a primer in an amplification reaction, theminimum size of such an oligonucleotide is the size required for theformation of a stable hybrid between the oligonucleotide and acomplementary sequence on a target nucleic acid molecule. Preferably,the oligonucleotides are at least 15 nucleotides, more preferably atleast 18 nucleotides, more preferably at least 19 nucleotides, morepreferably at least 20 nucleotides, even more preferably at least 25nucleotides in length.

Polynucleotides used as a probe are typically conjugated with adetectable label such as a radioisotope, hapten, an enzyme, biotin, afluorescent molecule or a chemiluminescent molecule. Oligonucleotides ofthe invention are useful in methods of detecting an allele of an SSII orother gene linked to a trait of interest, for example modified starch orfructan levels. Such methods, for example, employ nucleic acidhybridization and in many instances include oligonucleotide primerextension by a suitable polymerase (as used in PCR).

A variant of an oligonucleotide of the invention includes molecules ofvarying sizes of, and/or are capable of hybridising, for example, to thecereal genome close to that of, the specific oligonucleotide moleculesdefined herein. For example, variants may comprise additionalnucleotides (such as 1, 2, 3, 4, or more), or less nucleotides as longas they still hybridise to the target region. Furthermore, a fewnucleotides may be substituted without negatively influencing theability of the oligonucleotide to hybridise to the target region. Inaddition, variants may readily be designed which hybridise close to, forexample to within 50 nucleotides, the region of the plant genome wherethe specific oligonucleotides defined herein hybridise. Probes,oligonucleotides and the like are based upon the herein describedsequences or corrected versions thereof or variants thereof orfunctional homologs from other cereal plants.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides or their complementary forms displaying substantialsequence identity with a reference polynucleotide sequence. These termsalso encompass polynucleotides that are distinguished from a referencepolynucleotide by the addition, deletion or substitution of at least onenucleotide. Accordingly, the terms “polynucleotide variant” and“variant” include polynucleotides in which one or more nucleotides havebeen added or deleted, or replaced with different nucleotides. In thisregard, it is well understood in the art that certain alterationsinclusive of mutations, additions, deletions and substitutions can bemade to a reference polynucleotide whereby the altered polynucleotideretains the biological function or activity of the referencepolynucleotide. Accordingly, these terms encompass polynucleotides thatencode polypeptides that exhibit enzymatic or other regulatory activity,or polynucleotides capable of serving as selective probes or otherhybridising agents. In particular, this includes polynucleotides whichencode the same polypeptide or amino acid sequence but which vary innucleotide sequence by redundancy of the genetic code. The terms“polynucleotide variant” and “variant” also include naturally occurringallelic variants.

By “corresponds to” or “corresponding to” is meant a polynucleotide (a)having a nucleotide sequence that is substantially identical orcomplementary to all or most of a reference polynucleotide sequence or(b) encoding an amino acid sequence identical to an amino acid sequencein a peptide or protein. This phrase also includes within its scope apeptide or polypeptide having an amino acid sequence that issubstantially identical to a sequence of amino acids in a referencepeptide or protein. Terms used to describe sequence relationshipsbetween two or more polynucleotides or polypeptides include “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, “substantial identity” and “identical”, and aredefined with respect to a minimum number of nucleotides or amino acidresidues or over the full length. The terms “sequence identity” and“identity” are used interchangeably herein to refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

The % identity of a polynucleotide can be determined by GAP (Needlemanand Wunsch, J. Mol. Biol. 48: 443-453, 1970) analysis (GCG program) witha gap creation penalty=5, and a gap extension penalty=0.3. Unless statedotherwise, the query sequence is at least 45 nucleotides in length, andthe GAP analysis aligns the two sequences over a region of at least 45nucleotides. Preferably, the query sequence is at least 150 nucleotidesin length, and the GAP analysis aligns the two sequences over a regionof at least 150 nucleotides. More preferably, the query sequence is atleast 300 nucleotides in length and the GAP analysis aligns the twosequences over a region of at least 300 nucleotides, or at least 400, atleast 500 or at least 600 nucleotides in each case. Reference also maybe made to the BLAST family of programs as for example disclosed byAltschul et al., Nucleic Acids Res. 25: 3389, 1997. A detaileddiscussion of sequence analysis can be found in Unit 19.3 of Ausubel etal., “Current Protocols in Molecular Biology”, John Wiley & Sons Inc,1994-1998, Chapter 15.

Nucleotide or amino acid sequences are indicated as “essentiallysimilar” when such sequences have a sequence identity of at least 80%,particularly at least 85%, quite particularly at least 90%, especiallyat least 95%, more especially are identical. It is clear that when RNAsequences are described as essentially similar to, correspond to, orhave a certain degree of sequence identity with, DNA sequences, thymine(T) in the DNA sequence is considered equal to uracil (U) in the RNAsequence.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 80%, more preferably at least85%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, more preferably at least 93%, more preferablyat least 94%, more preferably at least 95%, more preferably at least96%, more preferably at least 97%, more preferably at least 98%, morepreferably at least 99%, more preferably at least 99.1%, more preferablyat least 99.2%, more preferably at least 99.3%, more preferably at least99.4%, more preferably at least 99.5%, more preferably at least 99.6%,more preferably at least 99.7%, more preferably at least 99.8%, and evenmore preferably at least 99.9% identical to the relevant nominated SEQID NO.

Preferably, a polynucleotide of the invention which encodes apolypeptide with SSII activity is greater than 800, preferably greaterthan 900, and even more preferably greater than 1,000 or 2000nucleotides in length.

Polynucleotides of the present invention may possess, when compared tonaturally occurring molecules, one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagenesis on the nucleic acid).

The present invention refers to the stringency of hybridizationconditions to define the extent of complementarity of twopolynucleotides. “Stringency” as used herein, refers to the temperatureand ionic strength conditions, and presence or absence of certainorganic solvents, during hybridization and washing. The higher thestringency, the higher will be the degree of complementarity between atarget nucleotide sequence and the labelled polynucleotide sequence(probe). “Stringent conditions” refers to temperature and ionicconditions under which only nucleotide sequences having a high frequencyof complementary bases will hybridize. As used herein, the term“hybridizes under low stringency, medium stringency, high stringency, orvery high stringency conditions” describes conditions for hybridizationand washing. Guidance for performing hybridization reactions can befound in Ausubel et al., (eds.), Current Protocols in Molecular Biology,John Wiley & Sons, NY, 6.3.1-6.3.6., 1989. Aqueous and nonaqueousmethods are described in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: 1) lowstringency hybridization conditions are for hybridization in 6× sodiumchloride/sodium citrate (SSC) at 45° C., followed by two washes in0.2×SSC, 0.1% SDS at 50-55° C.; 2) medium stringency hybridizationconditions are for hybridization in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringencyhybridization conditions are for hybridization in 6×SSC at 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and 4)very high stringency hybridization conditions are for hybridization in0.5 M sodium phosphate buffer, 7% SDS at 65° C., followed by one or morewashes at 0.2×SSC, 1% SDS at 65° C.

Polypeptides

The terms “polypeptide” and “protein” are generally usedinterchangeably. The terms “proteins” and “polypeptides” as used hereinalso include variants, mutants, modifications, analogs and/orderivatives of the polypeptides of the invention as described herein. Asused herein, “substantially purified polypeptide” refers to apolypeptide that has been separated from the lipids, nucleic acids,other peptides and other molecules with which it is associated in itsnative state. Preferably, the substantially purified polypeptide is atleast 90% free from other components with which it is naturallyassociated. By “recombinant polypeptide” is meant a polypeptide madeusing recombinant techniques, i.e., through the expression of arecombinant polynucleotide in a cell, preferably a plant cell and morepreferably a cereal plant cell.

Illustrative polypeptides having SSII activity are set out in thesequence listing and described in Table 7. Accordingly, the presentinvention proposes without limitation the modification of SSIIpolypeptides having the amino acid sequences set forth in SEQ ID NO: 2,4, 6, 8, 11, 13, 17, 19, 21, and 23 and naturally occurring variants,corrected versions thereof and variants as described herein such asvariants having about 80% sequence identity.

With regard to a defined polypeptide, it will be appreciated that %identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 75%, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, more preferablyat least 93%, more preferably at least 94%, more preferably at least95%, more preferably at least 96%, more preferably at least 97%, morepreferably at least 98%, more preferably at least 99%, more preferablyat least 99.1%, more preferably at least 99.2%, more preferably at least99.3%, more preferably at least 99.4%, more preferably at least 99.5%,more preferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

The % identity of a polypeptide relative to another polypeptide can bedetermined by GAP (Needleman and Wunsch, 1970 (supra)) analysis (GCGprogram) with a gap creation penalty=5, and a gap extension penalty=0.3.The query sequence is at least 15 amino acids in length, and the GAPanalysis aligns the two sequences over a region of at least 15 aminoacids. More preferably, the query sequence is at least 50 amino acids inlength, and the GAP analysis aligns the two sequences over a region ofat least 50 amino acids. More preferably, the query sequence is at least100 amino acids in length and the GAP analysis aligns the two sequencesover a region of at least 100 amino acids. Even more preferably, thequery sequence is at least 250 amino acids in length and the GAPanalysis aligns the two sequences over a region of at least 250 aminoacids.

As used herein a “biologically active” fragment of a polypeptide is aportion of a polypeptide of the invention, less than full length, whichmaintains a defined activity of the full-length polypeptide. In aparticularly preferred embodiment, the biologically active fragment isable to synthesize starch to produce amylose chains having a DP of atleast 15. Biologically active fragments can be any size as long as theymaintain the defined activity, but are preferably at least 200 or atleast 250 amino acid residues long.

With regard to a defined polypeptide, it will be appreciated that %identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, more preferably at least 93%, more preferablyat least 94%, more preferably at least 95%, more preferably at least96%, more preferably at least 97%, more preferably at least 98%, morepreferably at least 99%, more preferably at least 99.1%, more preferablyat least 99.2%, more preferably at least 99.3%, more preferably at least99.4%, more preferably at least 99.5%, more preferably at least 99.6%,more preferably at least 99.7%, more preferably at least 99.8%, and evenmore preferably at least 99.9% identical to the relevant nominated SEQID NO.

Amino acid sequence mutants of the polypeptides of the present inventioncan be prepared by introducing appropriate nucleotide changes into anucleic acid of the present invention, or by in vitro synthesis of thedesired polypeptide. Such mutants include, for example, deletions,insertions or substitutions of residues within the amino acid sequence.A combination of deletion, insertion and substitution can be made toarrive at the final construct, provided that the final peptide productpossesses the desired characteristics.

Mutant (altered) peptides can be prepared using any technique known inthe art. For example, a polynucleotide of the invention can be subjectedto in vitro mutagenesis. Such in vitro mutagenesis techniques includesub-cloning the polynucleotide into a suitable vector, transforming thevector into a “mutator” strain such as the E. coli XL-1 red (Stratagene)and propagating the transformed bacteria for a suitable number ofgenerations. In another example, the polynucleotides of the inventionare subjected to DNA shuffling techniques as broadly described byHarayama, Trends Biotechnol. 16: 76-82, 1998. These DNA shufflingtechniques may include genes related to those of the present invention,such as SSII genes from plant species other than wheat or barley, and/orinclude different genes from the same plant encoding similar proteins,such as the wheat or barley starch synthase I or III genes. Productsderived from mutated/altered DNA can readily be screened usingtechniques described herein to determine if they possess, for example,starch synthase activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active site(s). Other sites of interestare those in which particular residues obtained from various strains orspecies are identical. These positions may be important for biologicalactivity. These sites, especially those falling within a sequence of atleast three other identically conserved sites, are preferablysubstituted in a relatively conservative manner. Such conservativesubstitutions are shown in Table 9 under the heading of “exemplarysubstitutions”.

Polypeptides of the present invention can be produced in a variety ofways, including production and recovery of natural polypeptides,production and recovery of recombinant polypeptides, and chemicalsynthesis of the polypeptides. In one embodiment, an isolatedpolypeptide of the present invention is produced by culturing a cellcapable of expressing the polypeptide under conditions effective toproduce the polypeptide, and recovering the polypeptide. A preferredcell to culture is a recombinant cell of the present invention.Effective culture conditions include, but are not limited to, effectivemedia, bioreactor, temperature, pH and oxygen conditions that permitpolypeptide production. An effective medium refers to any medium inwhich a cell is cultured to produce a polypeptide of the presentinvention. Such medium typically comprises an aqueous medium havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells ofthe present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes, and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

The present invention refers to elements which are operably connected orlinked. “Operably connected” or “operably linked” and the like refer toa linkage of polynucleotide elements in a functional relationship.Typically, operably connected nucleic acid sequences are contiguouslylinked and, where necessary to join two protein coding regions,contiguous and in reading frame. A coding sequence is “operablyconnected to” another coding sequence when RNA polymerase willtranscribe the two coding sequences into a single RNA, which iftranslated is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein, the term “cis-acting sequence”, “cis-acting element” or“cis-regulatory region” or “regulatory region” or similar term shall betaken to mean any sequence of nucleotides, which when positionedappropriately and connected relative to an expressible genetic sequence,is capable of regulating, at least in part, the expression of thegenetic sequence. Those skilled in the art will be aware that acis-regulatory region may be capable of activating, silencing,enhancing, repressing or otherwise altering the level of expressionand/or cell-type-specificity and/or developmental specificity of a genesequence at the transcriptional or post-transcriptional level. Incertain embodiments of the present invention, the cis-acting sequence isan activator sequence that enhances or stimulates the expression of anexpressible genetic sequence.

“Operably connecting” a promoter or enhancer element to a transcribablepolynucleotide means placing the transcribable polynucleotide (e.g.,protein-encoding polynucleotide or other transcript) under theregulatory control of a promoter, which then controls the transcriptionof that polynucleotide. In the construction of heterologouspromoter/structural gene combinations, it is generally preferred toposition a promoter or variant thereof at a distance from thetranscription start site of the transcribable polynucleotide which isapproximately the same as the distance between that promoter and theprotein coding region it controls in its natural setting; i.e., the genefrom which the promoter is derived. As is known in the art, somevariation in this distance can be accommodated without loss of function.Similarly, the preferred positioning of a regulatory sequence element(e.g., an operator, enhancer etc) with respect to a transcribablepolynucleotide to be placed under its control is defined by thepositioning of the element in its natural setting; i.e., the genes fromwhich it is derived.

“Promoter” or “promoter sequence” as used herein refers to a region of agene, generally upstream (5′) of the RNA encoding region, which controlsthe initiation and level of transcription in the cell of interest. A“promoter” includes the transcriptional regulatory sequences of aclassical genomic gene, including a TATA box and CCAAT box sequences, aswell as additional regulatory elements (i.e., upstream activatingsequences, enhancers and silencers) that alter gene expression inresponse to developmental and/or environmental stimuli, or in atissue-specific or cell-type-specific manner. A promoter is usually, butnot necessarily (for example, some PolIII promoters), positionedupstream of a structural gene, the expression of which it regulates.Furthermore, the regulatory elements comprising a promoter are usuallypositioned within 2 kb of the start site of transcription of the gene.Promoters may contain additional specific regulatory elements, locatedmore distal to the start site to further enhance expression in a cell,and/or to alter the timing or inducibility of expression of a structuralgene to which it is operably connected.

“Constitutive promoter” refers to a promoter that directs expression ofan operably linked transcribed sequence in many or all tissues of aplant. The term constitutive as used herein does not necessarilyindicate that a gene is expressed at the same level in all cell types,but that the gene is expressed in a wide range of cell types, althoughsome variation in level is often detectable. “Selective expression” asused herein refers to expression almost exclusively in specific organsof the plant, such as, for example, endosperm, embryo, leaves, fruit,tubers or root. In one embodiment, a promoter is expressed in allphotosynthetic tissue, which may correspond to all aerial parts of theplant, for example a promoter that is involved in expressing a generequired for photosynthesis such as rubisco small subunit promoters. Theterm may also refer to expression at specific developmental stages in anorgan, such as in early or late embryogenesis or different stages ofmaturity; or to expression that is inducible by certain environmentalconditions or treatments. Selective expression may therefore becontrasted with constitutive expression, which refers to expression inmany or all tissues of a plant under most or all of the conditionsexperienced by the plant.

Selective expression may also result in compartmentation of the productsof gene expression in specific plant tissues, organs or developmentalstages. Compartmentation in specific subcellular locations such as theendosperm, cytosol, vacuole, or apoplastic space may be achieved by theinclusion in the structure of the gene product of appropriate signalsfor transport to the required cellular compartment, or in the case ofthe semi-autonomous organelles (plastids and mitochondria) byintegration of the transgene with appropriate regulatory sequencesdirectly into the organelle genome.

A “tissue-specific promoter” or “organ-specific promoter” is a promoterthat is preferentially expressed in one tissue or organ relative to manyother tissues or organs, preferably most if not all other tissues ororgans in a plant. Typically, the promoter is expressed at a level10-fold higher in the specific tissue or organ than in other tissues ororgans. An illustrative tissue specific promoter is the promoter forhigh molecular weight (HMW) glutenin gene, Bx17 which is expressedpreferentially in the developing endosperm of cereal plants. Furtherendosperm specific promoters include the high molecular weight gluteninpromoter, the wheat SSI promoter, and the wheat BEII promoter.

The promoters contemplated by the present invention may be native to thehost plant to be transformed or may be derived from an alternativesource, where the region is functional in the host plant. Other sourcesinclude the Agrobacterium T-DNA genes, such as the promoters of genesfor the biosynthesis of nopaline, octapine, mannopine, or other opinepromoters; promoters from plants, such as ubiquitin promoters such asthe Ubi promoter from the maize ubi-1 gene, Christensen et al.,Transgen. Res., 5: 213-218, 1996 (see, e.g., U.S. Pat. No. 4,962,028) oractin promoters; tissue specific promoters (see, e.g., U.S. Pat. No.5,459,252 to Conkling et al.; WO 91/13992 to Advanced Technologies);promoters from viruses (including host specific viruses), or partiallyor wholly synthetic promoters. Numerous promoters that are functional inmono- and dicotyledonous plants are well known in the art (see, forexample, Greve, J. Mol. Appl. Genet., 1: 499-511, 1983; Salomon et al.,EMBO 1, 3: 141-146, 1984; Garfinkel et al., Cell, 27: 143-153, 1983;Barker et al., Plant Mol. Biol., 2: 235-350, 1983; including variouspromoters isolated from plants and viruses such as the cauliflowermosaic virus promoter (CaMV 35S, 19S). Many tissue specific promoterregions are known. Other transcriptional initiation regions whichpreferentially provide for transcription in certain tissues or undercertain growth conditions, include those from genes encoding napin, seedACP, zein, or other seed storage proteins. Fruit specific promoters arealso known, one such promoter is the E8 promoter, described by Deikmanet al., EMBO J., 2: 3315-3320, 1998 and DellaPenna et al., Plant Cell,1: 53-63, 1989. Non-limiting methods for assessing promoter activity aredisclosed by Medberry et al., Plant Cell, 4: 185-192, 1992; Medberry etal., Plant J. 3: 619-626, 1993, Sambrook et al., Molecular Cloning: ALaboratory Manual (2nd ed.). Cold Spring Harbour Laboratory, Cold SpringHarbour, N Y, 1989, and McPherson et al. (U.S. Pat. No. 5,164,316).

Alternatively or additionally, the promoter may be an inducible promoteror a developmentally regulated promoter which is capable of drivingexpression of the introduced polynucleotide at an appropriatedevelopmental stage of the plant. Other cis-acting sequences which maybe employed include transcriptional and/or translational enhancers.Enhancer regions are well known to persons skilled in the art, and caninclude an ATG translational initiation codon and adjacent sequences.The initiation codon must be in phase with the reading frame of thecoding sequence relating to the foreign or exogenous polynucleotide toensure translation of the entire sequence. The translation controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. Translational initiation regions may be providedfrom the source of the transcriptional initiation region, or from aforeign or exogenous polynucleotide. The sequence can also be derivedfrom the source of the promoter selected to drive transcription, and canbe specifically modified so as to increase translation of the mRNA.

The nucleic acid construct of the present invention typically comprisesa 3′ non-translated sequence from about 50 to 1,000 nucleotide basepairs which may include a transcription termination sequence. A 3′non-translated sequence may contain a transcription termination signalwhich may or may not include a polyadenylation signal and any otherregulatory signals capable of effecting mRNA processing. Apolyadenylation signal is characterized by effecting the addition ofpolyadenylic acid tracts to the 3′ end of the mRNA precursor.Polyadenylation signals are commonly recognized by the presence ofhomology to the canonical form 5′ AATAAA-3′ although variations are notuncommon. Transcription termination sequences which do not include apolyadenylation signal include terminators for PolI or PolIII RNApolymerase which comprise a run of four or more thymidines. Examples ofsuitable 3′ non-translated sequences are the 3′ transcribednon-translated regions containing a polyadenylation signal from thenopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et al.,Nucl. Acid Res., 11: 369, 1983) and the terminator for the T7 transcriptfrom the octopine synthase gene of Agrobacterium tumefaciens.Alternatively, suitable 3′ non-translated sequences may be derived fromplant genes such as the 3′ end of the protease inhibitor I or II genesfrom potato or tomato, the soybean storage protein genes and the smallsubunit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene,although other 3′ elements known to those of skill in the art can alsobe employed. Alternatively, 3′ non-translated regulatory sequences canbe obtained de novo as, for example, described by An, Methods inEnzymology, 153: 292, 1987, which is incorporated herein by reference.

As the DNA sequence inserted between the transcription initiation siteand the start of the coding sequence, i.e., the untranslated 5′ leadersequence (5′UTR), can influence gene expression, one can also employ aparticular leader sequence. Suitable leader sequences include those thatcomprise sequences selected to direct optimum expression of the foreignor endogenous DNA sequence. For example, such leader sequences include apreferred consensus sequence which can increase or maintain mRNAstability and prevent inappropriate initiation of translation as forexample described by Joshi, Nucl. Acid Res. 15: 6643, 1987.

Additionally, targeting sequences may be employed to target the enzymeencoded by the foreign or exogenous polynucleotide to an intracellularcompartment, for example to the chloroplast, within plant cells or tothe extracellular environment. For example, a nucleic acid sequenceencoding a transit or signal peptide sequence may be operably linked toa sequence that encodes a chosen enzyme of the subject invention suchthat, when translated, the transit or signal peptide can transport theenzyme to a particular intracellular or extracellular destination, andcan then be optionally post-translationally removed. Transit or signalpeptides act by facilitating the transport of proteins throughintracellular membranes, e.g., endoplasmic reticulum, vacuole, vesicle,plastid, mitochondrial and plasmalemma membranes. For example, thetargeting sequence can direct a desired protein to a particularorganelle such as a vacuole or a plastid (e.g., a chloroplast), ratherthan to the cytosol. Thus, the nucleic acid construct of the inventioncan further comprise a plastid transit peptide-encoding nucleic acidsequence operably linked between a promoter region and the foreign orexogenous polynucleotide.

Vectors

The present invention includes use of vectors for manipulation ortransfer of genetic constructs. By “vector” is meant a nucleic acidmolecule, preferably a DNA molecule derived, for example, from aplasmid, bacteriophage, or plant virus, into which a nucleic acidsequence may be inserted or cloned. A vector preferably contains one ormore unique restriction sites and may be capable of autonomousreplication in a defined host cell including a target cell or tissue ora progenitor cell or tissue thereof, or be integrable with the genome ofthe defined host such that the cloned sequence is reproducible.Accordingly, the vector may be an autonomously replicating vector, i.e.,a vector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a linear orclosed circular plasmid, an extrachromosomal element, a minichromosome,or an artificial chromosome. The vector may contain any means forassuring self-replication. Alternatively, the vector may be one which,when introduced into a cell, is integrated into the genome of therecipient cell and replicated together with the chromosome(s) into whichit has been integrated. A vector system may comprise a single vector orplasmid, two or more vectors or plasmids, which together contain thetotal DNA to be introduced into the genome of the host cell, or atransposon. The choice of the vector will typically depend on thecompatibility of the vector with the cell into which the vector is to beintroduced. The vector may also include a selection marker such as anantibiotic resistance gene, a herbicide resistance gene or other genethat can be used for selection of suitable transformants. Examples ofsuch genes are well known to those of skill in the art.

The nucleic acid construct of the invention can be introduced into avector, such as a plasmid. Plasmid vectors typically include additionalnucleic acid sequences that provide for easy selection, amplification,and transformation of the expression cassette in prokaryotic andeukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors.Additional nucleic acid sequences include origins of replication toprovide for autonomous replication of the vector, selectable markergenes, preferably encoding antibiotic or herbicide resistance, uniquemultiple cloning sites providing for multiple sites to insert nucleicacid sequences or genes encoded in the nucleic acid construct, andsequences that enhance transformation of prokaryotic and eukaryotic(especially plant) cells.

By “marker gene” is meant a gene that imparts a distinct phenotype tocells expressing the marker gene and thus allows such transformed cellsto be distinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, i.e., by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). The marker gene and the nucleotide sequence ofinterest do not have to be linked.

To facilitate identification of transformants, the nucleic acidconstruct desirably comprises a selectable or screenable marker gene as,or in addition to, the foreign or exogenous polynucleotide. The actualchoice of a marker is not crucial as long as it is functional (i.e.,selective) in combination with the plant cells of choice. The markergene and the foreign or exogenous polynucleotide of interest do not haveto be linked, since co-transformation of unlinked genes as, for example,described in U.S. Pat. No. 4,399,216 is also an efficient process inplant transformation.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, kanamycin, erythromycin,chloramphenicol or tetracycline resistance. Exemplary selectable markersfor selection of plant transformants include, but are not limited to, ahyg gene which encodes hygromycin B resistance; a neomycinphosphotransferase (npt) gene conferring resistance to kanamycin,paromomycin, G418 and the like as, for example, described by Potrykus etal., Mol. Gen. Genet. 199: 183, 1985; a glutathione-S-transferase genefrom rat liver conferring resistance to glutathione derived herbicidesas, for example, described in EP-A 256223; a glutamine synthetase geneconferring, upon overexpression, resistance to glutamine synthetaseinhibitors such as phosphinothricin as, for example, described in WO87/05327, an acetyltransferase gene from Streptomyces viridochromogenesconferring resistance to the selective agent phosphinothricin as, forexample, described in EP-A 275957, a gene encoding a5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance toN-phosphonomethylglycine as, for example, described by Hinchee et al.,Biotech. 6: 915, 1988, a bar gene conferring resistance againstbialaphos as, for example, described in WO 91/02071; a nitrilase genesuch as bxn from Klebsiella ozaenae which confers resistance tobromoxynil (Stalker et al., Science, 242: 419, 1988); a dihydrofolatereductase (DHFR) gene conferring resistance to methotrexate (Thillet etal., J. Biol. Chem. 263: 12500, 1988); a mutant acetolactate synthasegene (ALS), which confers resistance to imidazolinone, sulfonylurea orother ALS-inhibiting chemicals (EP-A-154 204); a mutated anthranilatesynthase gene that confers resistance to 5-methyl tryptophan; or adalapon dehalogenase gene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known, a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known, an aequorin gene(Prasher et al., Biochem. Biophys. Res. Comm. 126: 1259-68, 1985), whichmay be employed in calcium-sensitive bioluminescence detection; a greenfluorescent protein gene (Niedz et al., Plant Cell Reports, 14: 403,1995); a luciferase (luc) gene (Ow et al., Science, 234: 856, 1986),which allows for bioluminescence detection, and others known in the art.By “reporter molecule” as used in the present specification is meant amolecule that, by its chemical nature, provides an analyticallyidentifiable signal that facilitates determination of promoter activityby reference to protein product.

Methods of Modifying Gene Expression

The level of a protein, for example an enzyme involved in starchsynthesis in developing endosperm of a cereal plant, may be modulated byincreasing the level of expression of a nucleotide sequence that codesfor the protein in a plant cell, or decreasing the level of expressionof a gene encoding the protein in the plant, leading to altered fructanaccumulation in grain. The level of expression of a gene may bemodulated by altering the copy number per cell, for example byintroducing a synthetic genetic construct comprising the coding sequenceand a transcriptional control element that is operably connected theretoand that is functional in the cell. A plurality of transformants may beselected and screened for those with a favourable level and/orspecificity of transgene expression arising from influences ofendogenous sequences in the vicinity of the transgene integration site.A favourable level and pattern of transgene expression is one whichresults in a substantial increase in fructan levels. This may bedetected by simple testing of grain from the transformants.Alternatively, a population of mutagenized grain or a population ofplants from a breeding program may be screened for individual lines withaltered fructan accumulation.

Reducing gene expression may be achieved through introduction andtranscription of a “gene-silencing chimeric gene” introduced into theplant cell. The gene-silencing chimeric gene may be introduced stablyinto the plant cell's genome, preferably nuclear genome, or it may beintroduced transiently, for example on a viral vector. As used herein“gene-silencing effect” refers to the reduction of expression of atarget nucleic acid in a a plant cell, which can be achieved byintroduction of a silencing RNA. Such reduction may be the result ofreduction of transcription, including via methylation of chromatinremodeling, or post-transcriptional modification of the RNA molecules,including via RNA degradation, or both. Gene-silencing includes anabolishing of the expression of the target nucleic acid or gene and apartial effect in either extent or duration. It is sufficient that thelevel of expression of the target nucleic acid in the presence of thesilencing RNA is lower that in the absence thereof. The level ofexpression may be reduced by at least about 40%, or at least about 50%,or at least about 60%, or at least about 70%, or at least about 80%, orat least about 90%, or at least about 95%, or at least about 99%. Thetarget nucleic acid may be a gene involved in starch synthesis ormetabolism, for example starch degradation, but may also include anyother endogenous genes, transgenes or exogenous genes such as viralgenes which may not be present in the plant cell at the time ofintroduction of the transgene.

Antisense RNA Molecules

Antisense techniques may be used to reduce gene expression according tothe invention. The term “antisense RNA” shall be taken to mean an RNAmolecule that is complementary to at least a portion of a specific mRNAmolecule and capable of reducing expression of the gene encoding themRNA. Such reduction typically occurs in a sequence-dependent manner andis thought to occur by interfering with a post-transcriptional eventsuch as mRNA transport from nucleus to cytoplasm, mRNA stability orinhibition of translation. The use of antisense methods is well known inthe art (see for example, Hartmann and Endres, Manual of AntisenseMethodology, Kluwer, 1999). The use of antisense techniques in plantshas been reviewed by Bourque, Plant Sci. 105: 125-149, 1995 and Senior,Biotech. Genet. Engin. Revs. 15: 79-119, 1998. Bourque, 1995 (supra)lists a large number of examples of how antisense sequences have beenutilized in plant systems as a method of gene inactivation. She alsostates that attaining 100% inhibition of any enzyme activity may not benecessary as partial inhibition will more than likely result inmeasurable change in the system. Senior, 1998 (supra) states thatantisense methods are now a very well established technique formanipulating gene expression.

As used herein, the term “an antisense polynucleotide which hybridisesunder physiological conditions” means that the polynucleotide (which isfully or partially single stranded) is at least capable of forming adouble stranded polynucleotide with an RNA product of the gene to beinhibited, typically the mRNA encoding a protein such as those providedherein, under normal conditions in a cell. Antisense molecules mayinclude sequences that correspond to the structural genes or forsequences that effect control over the gene expression or splicingevent. For example, the antisense sequence may correspond to the codingregion of the targeted gene, or the 5′-untranslated region (UTR) or the3′-UTR or combination of these. It may be complementary in part tointron sequences, which may be spliced out during or aftertranscription, but is preferably complementary only to exon sequences ofthe target gene. In view of the generally greater divergence of theUTRs, targeting these regions provides greater specificity of geneinhibition.

The length of the antisense sequence should be at least 19 contiguousnucleotides, preferably at least 25 or 30 or 50 nucleotides, and morepreferably at least 100, 200, 500 or 1000 nucleotides, to a maximum ofthe full length of the gene to be inhibited. The full-length sequencecomplementary to the entire gene transcript may be used. The length ismost preferably 100-2000 nucleotides. The degree of identity of theantisense sequence to the targeted transcript should be at least 90% andmore preferably 95-100%. The antisense RNA molecule may of coursecomprise unrelated sequences which may function to stabilize themolecule.

Genetic constructs to express an antisense RNA may be readily made byjoining a promoter sequence to a region of the target gene in an“antisense” orientation, which as used herein refers to the reverseorientation relative to the orientation of transcription and translation(if it occurs) of the sequence in the target gene in the plant cell.Accordingly, also provided by this invention is a nucleic acid moleculesuch as a chimeric DNA coding for an antisense RNA of the invention,including cells, tissues, organs, plants, grain and the like comprisingthe nucleic acid molecule.

Ribozymes

The term “ribozyme” as used herein refers to an RNA molecule whichspecifically recognizes a distinct substrate RNA and catalyzes itscleavage. Typically, the ribozyme contains a region of nucleotides whichare complementary to a region of the target RNA, enabling the ribozymeto specifically hybridize to the target RNA under physiologicalconditions, for example in the cell in which the ribozyme acts, and anenzymatic region referred to herein as the “catalytic domain”. The typesof ribozymes that are particularly useful in this invention are thehammerhead ribozyme (Haseloff and Gerlach, Nature 334: 585-591, 1988;Perriman et al., Gene, 113: 157-163, 1992) and the hairpin ribozyme(Shippy et al., Mol. Biotech. 12: 117-129, 1999). DNA encoding theribozymes can be synthesized using methods well known in the art and maybe incorporated into a genetic construct or expression vector forexpression in the cell of interest. Accordingly, also provided by thisinvention is a nucleic acid molecule such as a chimeric DNA coding for aribozyme of the invention, including cells, tissues, organs, plants,grain and the like comprising the nucleic acid molecule. Typically, theDNA encoding the ribozyme is inserted into an expression cassette undercontrol of a promoter and a transcription termination signal thatfunction in the cell. Specific ribozyme cleavage sites within anypotential RNA target may be identified by scanning the target moleculefor ribozyme cleavage sites which include the trinucleotide sequencesGUA, GUU and GUC. Once identified, short RNA sequences of between about5 and 20 ribonucleotides corresponding to the region of the target gene5′ and 3′ of the cleavage site may be evaluated for predicted structuralfeatures such as secondary structure that may render the oligonucleotidesequence less suitable. When employed, ribozymes may be selected fromthe group consisting of hammerhead ribozymes, hairpin ribozymes, axeheadribozymes, newt satellite ribozymes, Tetrahymena ribozymes and RNAse Pribozymes, and are designed according to methods known in the art basedon the sequence of the target gene (for instance, see U.S. Pat. No.5,741,679). The suitability of candidate targets may also be evaluatedby testing their accessibility to hybridization with complementaryoligonucleotides, using ribonuclease protection assays.

As with antisense polynucleotides described herein, ribozymes of theinvention should be capable of hybridizing to a target nucleic acidmolecule (for example an mRNA encoding a polypeptide provided as SEQ IDNO:2, SEQ ID NO:4) under “physiological conditions”, namely thoseconditions within a cell, especially conditions in a plant cell such asa wheat or barley cell.

RNA Interference/Duplex RNA

As used herein, “artificially introduced dsRNA molecule” refers to theintroduction of dsRNA molecule, which may e.g. occur endogenously bytranscription from a chimeric gene encoding such dsRNA molecule, howeverdoes not refer to the conversion of a single stranded RNA molecule intoa dsRNA inside the eukaryotic cell or plant cell. RNA interference(RNAi) is particularly useful for specifically reducing the expressionof a gene or inhibiting the production of a particular protein. Althoughnot wishing to be limited by theory, Waterhouse et al., Proc. Natl.Acad. Sci. U.S.A. 95: 13959-13964, 1998 have provided a model for themechanism by which dsRNA can be used to reduce protein production. Thistechnology relies on the presence of dsRNA molecules that contain asequence that is essentially identical to the mRNA of the gene ofinterest or part thereof. Conveniently, the dsRNA can be produced from asingle promoter in a recombinant vector or host cell, where the senseand anti-sense sequences are transcribed to produce a hairpin RNA inwhich the sense and anti-sense sequences hybridize to form the dsRNAregion with an intervening sequence or spacer region forming a loopstructure, so the hairpin RNA comprises a stem-loop structure. Thedesign and production of suitable dsRNA molecules for the presentinvention is well within the capacity of a person skilled in the art,particularly considering Waterhouse et al., 1998 (supra), Smith et al.,Nature, 407: 319-320, 2000, WO 99/53050, WO 99/49029, and WO 01/34815.Accordingly, also provided by this invention is a nucleic acid moleculesuch as a chimeric DNA coding for a duplex RNA such as a hairpin RNA ofthe invention, including cells, tissues, organs, plants, grain and thelike comprising the nucleic acid molecule.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded RNA product(s) with homology to the targetgene to be inactivated. The DNA therefore comprises both sense andantisense sequences that, when transcribed into RNA, can hybridize toform the double-stranded RNA region. In a preferred embodiment, thesense and antisense sequences are separated by a spacer region thatcomprises an intron which, when transcribed into RNA, is spliced out.This arrangement has been shown to result in a higher efficiency of genesilencing (Smith et al., 2000 (supra)). The double-stranded region maycomprise one or two RNA molecules, transcribed from either one DNAregion or two. The dsRNA may be classified as long hpRNA, having long,sense and antisense regions which can be largely complementary, but neednot be entirely complementary (typically forming a basepaired regionlarger than about 100 bp, preferably ranging between 200-1000 bp). hpRNAcan also be smaller with the double-stranded portion ranging in sizefrom about 30 to about 50 bp, or from 30 to about 100 bp (see WO04/073390, herein incorporated by reference). The presence of the doublestranded RNA region is thought to trigger a response from an endogenousplant system that processes the double stranded RNA to oligonucleotidesof 21-24 nucleotides long, and also uses these oligonucleotides forsequence-specific cleavage of the homologous RNA transcript from thetarget plant gene, efficiently reducing or eliminating the activity ofthe target gene.

The length of the sense and antisense sequences that hybridise shouldeach be at least 19 contiguous nucleotides, preferably at least 27 or 30or 50 nucleotides, and more preferably at least 100, 200, or 500nucleotides, up to the full-length sequence corresponding to the entiregene transcript. The lengths are most preferably 100-2000 nucleotides.The degree of identity of the sense and antisense sequences to thetargeted transcript should be at least 85%, preferably at least 90% andmore preferably 95-100%. The longer the sequence, the less stringent therequirement is for overall sequence identity. The RNA molecule may ofcourse comprise unrelated sequences which may function to stabilize themolecule. The RNA molecule may be a hybrid between different sequencestargeting different target RNAs, allowing reduction in expression ofmore than one target gene, or it may be one sequence which correspondsto a family of related target genes such as a multigene family. Thesequences used in the dsRNA preferably correspond to exon sequences ofthe target gene and may correspond to 5′ and/or 3′ untranslatedsequences or protein coding sequences or any combination thereof.

The promoter used to express the dsRNA-forming construct may be any typeof promoter if the resulting dsRNA is specific for a gene product in thecell lineage targeted for destruction. Alternatively, the promoter maybe lineage specific in that it is only expressed in cells of aparticular development lineage. This might be advantageous where someoverlap in homology is observed with a gene that is expressed in anon-targeted cell lineage. The promoter may also be inducible byexternally controlled factors, or by intracellular environmentalfactors. Typically, the RNA molecule is expressed under the control of aRNA polymerase II or RNA polymerase III promoter. Examples of the latterinclude tRNA or snRNA promoters.

Other silencing RNA may be “unpolyadenylated RNA” comprising at least 20consecutive nucleotides having at least 95% sequence identity to thecomplement of a nucleotide sequence of an RNA transcript of the targetgene, such as described in WO 01/12824 or U.S. Pat. No. 6,423,885 (bothdocuments herein incorporated by reference). Yet another type ofsilencing RNA is an RNA molecule as described in WO 03/076619 (hereinincorporated by reference) comprising at least 20 consecutivenucleotides having at least 95% sequence identity to the sequence of thetarget nucleic acid or the complement thereof, and further comprising alargely-double stranded region as described in WO 03/076619.

MicroRNA regulation is a specialized branch of the RNA silencing pathwaythat evolved towards gene regulation, diverging from conventionalRNAi/PTGS. MicroRNAs are a specific class of small RNAs that are encodedin gene-like elements organized in a characteristic partial invertedrepeat. When transcribed, microRNA genes give rise to partiallybasepaired stem-looped precursor RNAs from which the microRNAs aresubsequently processed. MicroRNAs are typically about 21 nucleotides inlength. The released miRNAs are incorporated into RISC-like complexescontaining a particular subset of Argonaute proteins that exertsequence-specific gene repression (see, for example, Millar andWaterhouse, Funct Integr Genomics, 5: 129-135, 2005; Pasquinelli et al.,Curr Opin Genet Develop 15: 200-205, 2005; Almeida and Allshire, TrendsCell Biol. 15: 251-258, 2005, herein incorporated by reference).

Cosuppression

Another molecular biological approach that may be used for specificallyreducing gene expression is co-suppression. The mechanism ofco-suppression is not well understood but is thought to involvepost-transcriptional gene silencing (PTGS) and in that regard may bevery similar to many examples of antisense suppression. It involvesintroducing an extra copy of a gene or a fragment thereof into a plantin the “sense orientation” with respect to a promoter for itsexpression, which as used herein refers to the same orientation astranscription and translation (if it occurs) of the sequence relative tothe sequence in the target gene. The size of the sense fragment, itscorrespondence to target gene regions, and its degree of homology to thetarget gene are as for the antisense sequences described above. In someinstances the additional copy of the gene sequence interferes with theexpression of the target plant gene. Reference is made to Patentspecification WO 97/20936 and European patent specification 0465572 formethods of implementing co-suppression approaches. The antisense,co-suppression or double stranded RNA molecules may also comprise alargely double-stranded RNA region, preferably comprising a nuclearlocalization signal, as described in WO 03/076619.

Any of these technologies for reducing gene expression can be used tocoordinately reduce the activity of multiple genes. For example, one RNAmolecule can be targeted against a family of related genes by targetinga region of the genes which is in common. Alternatively, unrelated genesmay be targeted by including multiple regions in one RNA molecule, eachregion targeting a different gene. This can readily be done by fusingthe multiple regions under the control of a single promoter.

Methods of Introducing Nucleic Acids into Plant Cells/Transformation

A number of techniques are available for the introduction of nucleicacid molecules into a plant host cell, well known to workers in the art.The term “transformation” means alteration of the genotype of anorganism, for example a bacterium or a plant, by the introduction of aforeign or exogenous nucleic acid. By “transformant” is meant anorganism so altered. As used herein the term “transgenic” refers to agenetically modified plant in which the endogenous genome issupplemented or modified by the integration, or stable maintenance in areplicable non-integrated form, of an introduced foreign or exogenousgene or sequence. By “transgene” is meant a foreign or exogenous gene orsequence that is introduced into the genome of a plant. The nucleic acidmolecule may be stably integrated into the genome of the plant, or itmay be replicated as an extrachromosomal element. By “genome” is meantthe total inherited genetic complement of the cell, plant or plant part,and includes chromosomal DNA, plastid DNA, mitochondrial DNA andextrachromosomal DNA molecules. The term “regeneration” as used hereinin relation to plant materials means growing a whole, differentiatedplant from a plant cell, a group of plant cells, a plant part such as,for example, from an embryo, scutellum, protoplast, callus, or othertissue, but not including growth of a plant from a seed.

The particular choice of a transformation technology will be determinedby its efficiency to transform certain plant species as well as theexperience and preference of the person practicing the invention with aparticular methodology of choice. It will be apparent to the skilledperson that the particular choice of a transformation system tointroduce a nucleic acid construct into plant cells is not essential toor a limitation of the invention, provided it achieves an acceptablelevel of nucleic acid transfer. Guidance in the practical implementationof transformation systems for plant improvement is provided by Birch,Ann Rev Plant Physiol Plant Mol Biol. 48: 297-326, 1997.

In principle, both dicotyledonous and monocotyledonous plants that areamenable to transformation can be modified by introducing a nucleic acidconstruct according to the invention into a recipient cell and growing anew plant that harbors and expresses a polynucleotide according to theinvention.

Introduction and expression of foreign or exogenous polynucleotides indicotyledonous plants such as tobacco, potato and legumes ormonocotyledonous plants such as cereals, including wheat, barley, rice,corn, oats, rye and sorghum has been shown to be possible using theT-DNA of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens(See, for example, Umbeck, U.S. Pat. No. 5,004,863, and Internationalapplication PCT/US93/02480). A construct of the invention may beintroduced into a plant cell utilizing A. tumefaciens containing the Tiplasmid. In using an A. tumefaciens culture as a transformation vehicle,it is most advantageous to use a non-oncogenic strain of theAgrobacterium as the vector carrier so that normal non-oncogenicdifferentiation of the transformed tissues is possible. It is preferredthat the Agrobacterium harbors a binary Ti plasmid system. Such a binarysystem comprises (1) a first Ti plasmid having a virulence regionessential for the introduction of transfer DNA (T-DNA) into plants, and(2) a chimeric plasmid. The chimeric plasmid contains at least oneborder region of the T-DNA region of a wild-type Ti plasmid flanking thenucleic acid to be transferred. Binary Ti plasmid systems have beenshown effective to transform plant cells as, for example, described byDe Framond, Biotechnology, 1: 262, 1983 and Hoekema et al., Nature, 303:179, 1983. Such a binary system is preferred inter alia because it doesnot require integration into the Ti plasmid in Agrobacterium.

Methods involving the use of Agrobacterium include, but are not limitedto: (a) co-cultivation of Agrobacterium with cultured isolatedprotoplasts; (b) transformation of plant cells or tissues withAgrobacterium; (c) transformation of seeds, apices or meristems withAgrobacterium, or (d) inoculation in planta such as the floral-dipmethod as described by Bechtold et al., C.R. Acad. Sci. Paris, 316:1194, 1993 or in wheat (as described in WO 00/63398, herein incorporatedby reference). This approach is based on the infiltration of asuspension of Agrobacterium cells. Alternatively, the chimeric constructmay be introduced using root-inducing (Ri) plasmids of Agrobacterium asvectors.

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example,Wan and Lemaux, Plant Physiol. 104: 37-48, 1994, Tingay et al., Plant J.11: 1369-1376, 1997, Canadian Patent Application No. 2,092,588,Australian Patent Application No. 61781/94, Australian Patent No.667939, U.S. Pat. No. 6,100,447, International Patent ApplicationPCT/US97/10621, U.S. Pat. Nos. 5,589,617, 6,541,257. Preferably,transgenic wheat, barley or other cereal plants are produced byAgrobacterium tumefaciens mediated transformation procedures. Vectorscarrying the desired nucleic acid construct may be introduced intoregenerable cereal cells of tissue cultured plants or explants. Theregenerable cells are preferably from the scutellum of immature embryos,mature embryos, callus derived from these, or the meristematic tissue.Immature embryos are preferably those from inflorescences about 10-15days after anthesis.

The genetic construct can also be introduced into plant cells byelectroporation as, for example, described by Fromm et al., Proc. Natl.Acad. Sci. U.S.A. 82: 5824, 1985 and Shimamoto et al., Nature, 338:274-276, 1989. In this technique, plant protoplasts are electroporatedin the presence of vectors or nucleic acids containing the relevantnucleic acid sequences. Electrical impulses of high field strengthreversibly permeabilize membranes allowing the introduction of nucleicacids. Electroporated plant protoplasts reform the cell wall, divide andform a plant callus.

Another method for introducing the nucleic acid construct into a plantcell is high velocity ballistic penetration by small particles (alsoknown as particle bombardment or microprojectile bombardment) with thenucleic acid to be introduced contained either within the matrix ofsmall beads or particles, or on the surface thereof as, for exampledescribed by Klein et al., Nature, 327: 70, 1987.

Alternatively, the nucleic acid construct can be introduced into a plantcell by contacting the plant cell using mechanical or chemical means.For example, a nucleic acid can be mechanically transferred bymicroinjection directly into plant cells by use of micropipettes.Alternatively, a nucleic acid may be transferred into the plant cell byusing polyethylene glycol which forms a precipitation complex withgenetic material that is taken up by the cell.

Mutagenesis

The plants of the invention can be produced and identified aftermutagenesis. This may provide a plant which is non-transgenic, which isdesirable in some markets.

Mutants can be either naturally occurring (that is to say, isolated froma natural source) or synthetic (for example, by performing mutagenesison the nucleic acid) or induced. Generally, a progenitor plant cell,tissue, seed or plant may be subjected to mutagenesis to produce singleor multiple mutations, such as nucleotide substitutions, deletions,additions and/or codon modification. In the context of this application,an “induced mutation” is an artificially induced genetic variation whichmay be the result of chemical, radiation or biologically-basedmutagenesis, for example transposon or T-DNA insertion. Preferredmutations are null mutations such as nonsense mutations, frameshiftmutations, insertional mutations or splice-site variants whichcompletely inactivate the gene. Nucleotide insertional derivativesinclude 5′ and 3′ terminal fusions as well as intra-sequence insertionsof single or multiple nucleotides. Insertional nucleotide sequencevariants are those in which one or more nucleotides are introduced intothe nucleotide sequence, which may be obtained by random insertion withsuitable screening of the resulting products. Deletional variants arecharacterised by the removal of one or more nucleotides from thesequence. Preferably, a mutant gene has only a single insertion ordeletion of a sequence of nucleotides relative to the wild-type gene.Substitutional nucleotide variants are those in which at least onenucleotide in the sequence has been removed and a different nucleotideinserted in its place. The preferred number of nucleotides affected bysubstitutions in a mutant gene relative to the wild-type gene is amaximum of ten nucleotides, more preferably a maximum of 9, 8, 7, 6, 5,4, 3, or 2, or only one nucleotide. Such a substitution may be “silent”in that the substitution does not change the amino acid defined by thecodon. Alternatively, conservative substituents are designed to alterone amino acid for another similar acting amino acid. Typicalconservative substitutions are those made in accordance with Table 9“Exemplary substitutions”.

The term “mutation” as used herein does not include silent nucleotidesubstitutions which do not affect the activity of the gene, andtherefore includes only alterations in the gene sequence which affectthe gene activity. The term “polymorphism” refers to any change in thenucleotide sequence including such silent nucleotide substitutions.

In a preferred embodiment, the plant comprises a deletion of at leastpart of a SSII gene or a frameshift or splice site variation in suchgene.

Mutagenesis can be achieved by chemical or radiation means, for exampleEMS or sodium azide (Zwar and Chandler, Planta 197: 39-48, 1995)treatment of seed, or gamma irradiation, well know in the art. Isolationof mutants may be achieved by screening mutagenised plants or seed. Forexample, a mutagenized population of cereal plants may be screened forlow SSII activity in the leaf or grain starch, mutation of the SSII geneby a PCR or heteroduplex based assay, or loss of the SSII protein byELISA. In a polyploid plant, screening is preferably done in a genotypethat already lacks one or two of the SSII activities, for example in awheat plant already mutant in the SSII genes on two of the threegenomes, so that a mutant entirely lacking the functional activity issought. Alternatively, the mutation may be identified using techniquessuch as “tilling” in a population mutagenised with an agent such as EMS(Slade and Knauf, Transgenic Res. 14: 109-115, 2005). Such mutations maythen be introduced into desirable genetic backgrounds by crossing themutant with a plant of the desired genetic background and performing asuitable number of backcrosses to cross out the originally undesiredparent background.

The mutation may have been introduced into the plant directly bymutagenesis or indirectly by crossing of two parental plants, one ofwhich comprised the introduced mutation. The modified plants such ascereal plants may be transgenic or non-transgenic. Using mutagenesis, anon-transgenic plant lacking the function of interest may be produced.The invention also extends to the grain or other plant parts producedfrom the plants and any propagating material of the plants that can beused to produce the plants with the desired characteristics, such ascultured tissue or cells. The invention clearly extends to methods ofproducing or identifying such plants or the grain produced by suchplants.

Plants of the invention can be produced using the process known asTILLING (Targeting Induced Local Lesions IN Genomes). In a first step,introduced mutations such as novel single base pair changes are inducedin a population of plants by treating cells, seeds, pollen or otherplant parts with a chemical mutagen or radiation, and then advancingplants to a generation where mutations will be stably inherited. DNA isextracted, and seeds are stored from all members of the population tocreate a resource that can be accessed repeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as CelI, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TILLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique. TILLING is further described in Slade andKnauf, 2005 (supra), and Henikoff et al., Plant Physiol. 135: 630-636,2004, herein incorporated by reference.

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., Plant J.37: 778-786, 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

Genetic Linkage

As used herein, the term “genetically linked” refers to a marker locusand a second locus being sufficiently close on a chromosome that theywill be inherited together in more than 50% of meioses, e.g., notrandomly. This definition includes the situation where the marker locusand second locus form part of the same gene. Furthermore, thisdefinition includes the situation where the marker locus comprises apolymorphism that is responsible for the trait of interest (in otherwords the marker locus is directly “linked” to the phenotype). Thus, thepercent of recombination observed between the loci per generation(centimorgans (cM)), will be less than 50. In particular embodiments ofthe invention, genetically linked loci may be 45, 35, 25, 15, 10, 5, 4,3, 2, or 1 or less cM apart on a chromosome. Preferably, the markers areless than 5 cM or 2 cM apart and most preferably about 0 cM apart.

As used herein, the “other genetic markers” may be any molecules whichare linked to a desired trait of a cereal plant such as wheat or barley.Such markers are well known to those skilled in the art and includemolecular markers linked to genes determining traits such diseaseresistance, yield, plant morphology, grain quality, other dormancytraits such as grain colour, gibberellic acid content in the seed, plantheight, flour colour and the like. Examples of such genes in wheat arestem-rust resistance genes Sr2 or Sr38, the stripe rust resistance genesYr10 or Yr17, the nematode resistance genes such as Cre1 and Cre3,alleles at glutenin loci that determine dough strength such as Ax, Bx,Dx, Ay, By and Dy alleles, the Rht genes that determine a semi-dwarfgrowth habit and therefore lodging resistance (Eagles et al., Aust. J.Agric. Res. 52: 1 349-1356, 2001; Langridge et al., Aust. J. Agric. Res.52: 1043-1077, 2001; Sharp et al., Aust J Agric Res 52: 1357-1366,2001).

Marker assisted selection is a well recognised method of selecting forheterozygous plants required when backcrossing with a recurrent parentin a classical breeding program. The population of plants in eachbackcross generation will be heterozygous for the gene of interestnormally present in a 1:1 ratio in a backcross population, and themolecular marker can be used to distinguish the two alleles of the gene.By extracting DNA from, for example, young shoots and testing with aspecific marker for the introgressed desirable trait, early selection ofplants for further backcrossing is made whilst energy and resources areconcentrated on fewer plants.

Any molecular biological technique known in the art which is capable ofdetecting alleles of an SSII or other gene can be used in the methods ofthe present invention. Such methods include, but are not limited to, theuse of nucleic acid amplification, nucleic acid sequencing, nucleic acidhybridization with suitably labeled probes, single-strand conformationalanalysis (SSCA), denaturing gradient gel electrophoresis (DGGE),heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalyticnucleic acid cleavage or a combination thereof (see, for example,Lemieux, Current Genomics, 1: 301-311, 2000; Langridge et al., 2001(supra)). The invention also includes the use of molecular markertechniques to detect polymorphisms linked to alleles of (for example) anSSII gene which confers altered fructan accumulation. Such methodsinclude the detection or analysis of restriction fragment lengthpolymorphisms (RFLP), RAPD, amplified fragment length polymorphisms(AFLP) and microsatellite (simple sequence repeat, SSR) polymorphisms.The closely linked markers can be obtained readily by methods well knownin the art, such as Bulked Segregant Analysis, as reviewed by Langridgeet al., 2001 (supra).

The “polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” or“set of primers” consisting of “upstream” and a “downstream” primer, anda catalyst of polymerization, such as a DNA polymerase, and typically athermally-stable polymerase enzyme. Methods for PCR are known in theart, and are taught, for example, in “PCR” (McPherson and Moller (Ed),BIOS Scientific Publishers Ltd, Oxford, 2000). PCR can be performed oncDNA obtained from reverse transcribing mRNA isolated from plant cellsexpressing an SSII gene or on genomic DNA isolated from a plant.

A primer is an oligonucleotide sequence that is capable of hybridisingin a sequence specific fashion to the target sequence and being extendedduring the PCR. Amplicons or PCR products or PCR fragments oramplification products are extension products that comprise the primerand the newly synthesized copies of the target sequences. Multiplex PCRsystems contain multiple sets of primers that result in simultaneousproduction of more than one amplicon. Primers may be perfectly matchedto the target sequence or they may contain internal mismatched basesthat can result in the introduction of restriction enzyme or catalyticnucleic acid recognition/cleavage sites in specific target sequences.Primers may also contain additional sequences and/or contain modified orlabelled nucleotides to facilitate capture or detection of amplicons.Repeated cycles of heat denaturation of the DNA, annealing of primers totheir complementary sequences and extension of the annealed primers withpolymerase result in exponential amplification of the target sequence.The terms target or target sequence or template refer to nucleic acidsequences which are amplified.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.(supra) and Sambrook et al. (supra). Sequencing can be carried out byany suitable method, for example, dideoxy sequencing, chemicalsequencing or variations thereof. Direct sequencing has the advantage ofdetermining variation in any base pair of a particular sequence.

Plants

The term “plant” as used herein as a noun refers to whole plants andrefers to any member of the Kingdom Plantae, but as used as an adjectiverefers to any substance which is present in, obtained from, derivedfrom, or related to a plant, such as for example, plant organs (e.g.leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plantcells and the like. Plantlets and germinated seeds from which roots andshoots have emerged are also included within the meaning of “plant”. Theterm “plant parts” as used herein refers to one or more plant tissues ororgans which are obtained from a plant and which comprises genomic DNAof the plant. Plant parts include vegetative structures (for example,leaves, stems), roots, floral organs/structures, seed (including embryo,endosperm, and seed coat), plant tissue (for example, vascular tissue,ground tissue, and the like), cells and progeny of the same. The term“plant cell” as used herein refers to a cell obtained from a plant or ina plant and includes protoplasts or other cells derived from plants,gamete-producing cells, and cells which regenerate into whole plants.Plant cells may be cells in culture. By “plant tissue” is meantdifferentiated tissue in a plant or obtained from a plant (“explant”) orundifferentiated tissue derived from immature or mature embryos, seeds,roots, shoots, fruits, tubers, pollen, tumor tissue, such as crowngalls, and various forms of aggregations of plant cells in culture, suchas calli. Exemplary plant tissues in or from seeds are endosperm,scutellum, aleurone layer and embryo. The invention accordingly includesplants and plant parts and products comprising these, particularly graincomprising fructan.

As used herein, the term “grain” refers to mature seed of a plant, suchas is typically harvested commercially in the field. Mature cereal grainsuch as wheat or barley commonly has a moisture content of less thanabout 18-20%.

A “transgenic plant” as used herein refers to a plant that contains agene construct not found in a wild-type plant of the same species,variety or cultivar. That is, transgenic plants (transformed plants)contain genetic material (a transgene) that they did not contain priorto the transformation. The transgene may include genetic sequencesobtained from or derived from a plant cell, or another plant cell, or anon-plant source, or a synthetic sequence. Typically, the transgene hasbeen introduced into the plant by human manipulation such as, forexample, by transformation but any method can be used as one of skill inthe art recognizes. The genetic material is preferably stably integratedinto the genome of the plant. The introduced genetic material maycomprise sequences that naturally occur in the same species but in arearranged order or in a different arrangement of elements, for examplean antisense sequence. Plants containing such sequences are includedherein in “transgenic plants”. A “non-transgenic plant” is one which hasnot been genetically modified by the introduction of genetic material byrecombinant DNA techniques. In a preferred embodiment, the transgenicplants are homozygous for each and every gene that has been introduced(transgene) so that their progeny do not segregate for the desiredphenotype.

As used herein, the term “corresponding non-transgenic plant” refers toa plant which is isogenic relative to the transgenic plant but withoutthe transgene of interest. Preferably, the corresponding non-transgenicplant is of the same cultivar or variety as the progenitor of thetransgenic plant of interest, or a sibling plant line which lacks theconstruct, often termed a “segregant”, or a plant of the same cultivaror variety transformed with an “empty vector” construct, and may be anon-transgenic plant. “Wild type”, as used herein, refers to a cell,tissue or plant that has not been modified according to the invention.Wild-type cells, tissue or plants may be used as controls to comparelevels of expression of an exogenous nucleic acid or the extent andnature of trait modification with cells, tissue or plants modified asdescribed herein.

Transgenic plants, as defined in the context of the present inventioninclude progeny of the plants which have been genetically modified usingrecombinant techniques, wherein the progeny comprise the transgene ofinterest. Such progeny may be obtained by self-fertilisation of theprimary transgenic plant or by crossing such plants with another plantof the same species. This would generally be to modulate the productionof at least one protein/enzyme defined herein in the desired plant orplant organ. Transgenic plant parts include all parts and cells of saidplants comprising the transgene such as, for example, cultured tissues,callus and protoplasts.

Any of several methods may be employed to determine the presence of atransgene in a transformed plant. For example, polymerase chain reaction(PCR) may be used to amplify sequences that are unique to thetransformed plant, with detection of the amplified products by gelelectrophoresis or other methods. DNA may be extracted from the plantsusing conventional methods and the PCR reaction carried out usingprimers to amplify a specific DNA, the presence of which willdistinguish the transformed and non-transformed plants. For example,primers may be designed that will amplify a region of DNA from thetransformation vector reading into the construct and the reverse primerdesigned from the gene of interest. These primers will only amplify afragment if the plant has been successfully transformed. An alternativemethod to confirm a positive transformant is by Southern blothybridization, well known in the art. Plants which are transformed mayalso be identified i.e. distinguished from non-transformed or wild-typeplants by their phenotype, for example conferred by the presence of aselectable marker gene, or conferred by the phenotype of altered fructancontent of the grain of the plant, or related phenotype such as alteredstarch synthase activity.

As used herein, “germination” refers to the emergence of the root tipfrom the seed coat after imbibition. “Germination rate” refers to thepercentage of seeds in a population which have germinated over a periodof time, for example 7 or 10 days, after imbibition. A population ofseeds can be assessed daily over several days to determine thegermination percentage over time. With regard to seeds of the presentinvention, as used herein the term “germination rate which issubstantially the same” means that the germination rate of thetransgenic seeds is at least 90%, that of isogenic wild-type seeds.

Plants provided by or contemplated for use in the practice of thepresent invention include angiosperms, including both monocotyledons anddicotyledons. In preferred embodiments, the plants of the presentinvention are crop plants (for example, cereals and pulses, maize,wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, orpea), or other legumes. The plants may be grown for production of edibleroots, tubers, leaves, stems, flowers or fruit. Preferably, the plant isa cereal plant. Examples of cereal plants include, but are not limitedto, wheat, barley, rice, maize (corn), sorghum, oats, and rye. In oneembodiment, the cereal plant is other than barley mutants M292, M342 orbarley plants comprising the same SSII mutation, described in WO02/37955, herein incorporated by reference, such as wheat, rice, maizeor sorghum.

As used herein, the term “wheat” refers to any species of the GenusTriticum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. As is understood in the art,hexaploid wheats such as bread wheat comprise three genomes which arecommonly designated the A, B and D genomes, while tetraploid wheats suchas durum wheat comprise two genomes commonly designated the A and Bgenomes. Each wheat genome comprises 7 pairs of chromosomes which may beobserved by cytological methods during meiosis and thus identified, asis well known in the art. Wheat includes “hexaploid wheat” which hasgenome organization of AABBDD, comprised of 42 chromosomes, and“tetraploid wheat” which has genome organization of AABB, comprised of28 chromosomes. Hexaploid wheat includes T. aestivum, T. spelta, Tmacha, T. compactum, T. sphaerococcum, T. vavilovii, and interspeciescross thereof. Tetraploid wheat includes T. durum (also referred toherein as durum wheat or Triticum turgidum ssp. durum), T. dicoccoides,T. dicoccum, T. polonicum, and interspecies cross thereof. In addition,the term “wheat” includes potential progenitors of hexaploid ortetraploid Triticum sp. such as T. uartu, T. monococcum or T. boeoticumfor the A genome, Aegilops speltoides for the B genome, and T. tauschii(also known as Aegilops squarrosa or Aegilops tauschii) for the Dgenome. A wheat cultivar for use in the present invention may belong to,but is not limited to, any of the above-listed species. Also encompassedare plants that are produced by conventional techniques using Triticumsp. as a parent in a sexual cross with a non-Triticum species (such asrye [Secale cereale]), including but not limited to Triticale.Preferably, the wheat plant is suitable for commercial production ofgrain, such as commercial varieties of hexaploid wheat or durum wheat,having suitable agronomic characteristics which are known to thoseskilled in the art.

As used herein, the term “barley” refers to any species of the GenusHordeum, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. It is preferred that the plantis of a Hordeum species which is commercially cultivated such as, forexample, a strain or cultivar or variety of Hordeum vulgare or suitablefor commercial production of grain.

Food Production

In another aspect, the invention provides cereal plants and grain, andproducts obtained therefrom comprising fructan from the grain, that isuseful for food or feed production, the grain having increased levels offructan compared to corresponding wild-type grain. Preferably the plantfrom which the grain is obtained has a reduced level of SSII activity inthe endosperm during development. The plant of the present invention isuseful for food production and in particular for commercial foodproduction. Such food production might include the making of flour,dough or other products that might be an ingredient in commercial foodproduction. In an embodiment which is desirable for use in foodproduction, the seed or grain of the plant has a fructan content that isincreased relative to the wild-type plant. The grain may have a level ofactivity of degradative enzymes, particularly of one or more amylasessuch as α-amylase or β-amylase, which is reduced by the presence of atransgene or an introduced mutation which reduces expression of a geneencoding such a degradative enzyme in the grain. Flour or dough fromsuch grain has desirable properties for baking or other food production.

The desired genetic background of the plant will include considerationsof agronomic yield and other characteristics. Such characteristics mightinclude whether it is desired to have a winter or spring types,agronomic performance, disease resistance and abiotic stress resistance.For Australian use, one might want to cross the altered fructan traitinto wheat cultivars such as Baxter, Kennedy, Janz, Frame, Rosella,Cadoux, Diamondbird or other commonly grown varieties. Other varietieswill be suited for other growing regions. It is preferred that theplant, preferably wheat, variety of the invention provide a yield notless than 80% of the corresponding wild-type variety in at least somegrowing regions, more preferably not less than 85% and even morepreferably not less than 90%. The yield can readily be measured incontrolled field trials.

In further embodiments, other desirable characteristics include thecapacity to mill the grain, in particular the grain hardness. Anotheraspect that might make a plant of higher value is the degree of fructanor starch extraction from the grain, the higher extraction rates beingmore useful, or the protein content, the ration of amylose toamylopectin, or the content of other non-starch polysaccharides such asβ-glucan which also contribute to the dietary fibre content of the grainproducts. Grain shape is also another feature that can impact on thecommercial usefulness of a plant, thus grain shape can have an impact onthe ease or otherwise with which the grain can be milled.

Starch is readily isolated from grain of the invention using standardmethods, for example the method of Schulman and Kammiovirta, Starch, 43:387-389, 1991. On an industrial scale, wet or dry milling can be used.Starch granule size is important in the starch processing industry wherethere is separation of the larger A granules from the smaller Bgranules.

Food Products

The invention also encompasses foods, beverages or pharmaceuticalpreparations produced with products, preferably those comprisingincreased fructan, obtained from the plants or grain of the invention.Such food production might include the making of processed grain,wholemeal, flour, dough or other products that might be an ingredient incommercial food production. The grain of the invention or productsderived therefrom containing fructan may be used in a variety of foodapplications for human consumption. As used herein, “humans” refers toHomo sapiens. The grain can be used readily in food processingprocedures and therefore the invention includes milled, ground, kibbled,pearled or rolled grain or products obtained from the processed or wholegrain of the plants of the invention, including flour. These productsmay be then used in various food products, for example farinaceousproducts such as breads, cakes, biscuits and the like or food additivessuch as thickeners or binding agents or to make drinks, noodles, pastaor quick soups. The grain or products derived from the grain of theinvention are particularly desired in breakfast cereals or as extrudedproducts. The fructan may be incorporated into fat or oil products suchas margarine or shortening, salad dressing, egg products such asmayonnaise, dairy products such as icecream, yogurt or cheese, cerealproducts such as corn or wheat flour, fruit juices, other foods or foodmaterials, or the fructan may be processed into beverages or foods suchas bread, cake, biscuits, breakfast cereals, pasta, noodles or sauces.Fructan is also useful as a low calorie sweetening product.

In bread, the ingredients comprising fructan which may be in the form offlour or wholemeal may substitute for 10% (w/w) or more of unalteredflour or wholemeal, preferably substituting at least 30% and even morepreferably at least 50% of the unaltered flour or wholemeal. Theformulation might therefore be, for example, flour 70 parts,high-fructan starch 30 parts, fat 2 parts, salt 2 parts, improver 1part, yeast 2.5 parts. The production of the bread may be by a rapiddough technique or other techniques as is known by those skilled in theart.

Alternatively, the high-fructan product of the invention may beincorporated into a farinaceous based pasta product. The amount offructan of the invention employed in the pasta composition may be in therange of 5-20% (w/w) based on the total weight of farinaceous materialmore particularly in the range of 10 to 20%. Suitable other farinaceousmaterials will readily be chosen by a person skilled in the art. Othermaterial may also be added to the composition for example dry or liquideggs (yolks, whites, or both) or high protein substances such as milkprotein or fish protein. Vitamins, minerals, calcium salts, amino acids,buffering agents such as disodium hydrogen phosphate, seasoning, gum,gluten or glyceryl monostearate may also be added.

Other parts of the plants of the invention that are edible may be usedas foods for human consumption or as feed for animal use. For example,leaves, stems, roots, tubers, fruit, pods or extracts or parts of thesecomprising cells of the invention from any of these may be used forhuman or animal consumption. Increased fructan content of the plants ofthe invention and parts thereof may provide advantages for use of thesematerials as animal feed such as, for example, as feed for pigs, cattle,horses, poultry such as chickens and other animals.

Methods

The products or compounds of the present invention can be formulated inpharmaceutic compositions which are prepared according to conventionalpharmaceutical compounding techniques. See, for example, Remington'sPharmaceutical Sciences, 18^(th) Ed. Mack Publishing, Company, Easton,Pa., U.S.A., 1990. The composition may contain the active agent orpharmaceutically acceptable derivative active agent. These compositionsmay comprise, in addition to one of the active substances, apharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials well known in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The carrier may take a wide variety of forms depending onthe form of preparation desired for administration.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, powders,suspensions or emulsions. In preparing the compositions in oral dosageform, any of the usual pharmaceutical media may be employed, such as,for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques.

The active agent is preferably administered in a therapeuticallyeffective amount. An “effective amount” includes an amount of fructan orfructan containing product to promote an improvement in indicators ofintestinal health or an improvement in indicators of severity of thecondition such as diabetes, obesity, heart disease, hypertension,constipation, osteoporesis and cancer. The actual amount administeredand the rate and time-course of administration will depend on the natureand severity of the condition being treated. Prescription of treatment,e.g. decisions on dosage, timing, etc. is within the responsibility ofgeneral practitioners or specialists and typically takes account of thedisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of techniques and protocols can be found inRemington's Pharmaceutical Sciences, (supra).

The food or beverage or pharmaceutical preparation may be packaged readyfor sale or in bulk form. The invention also provides methods ofpreparing the food, beverage or pharmaceutical preparation of theinvention, and recipes or instructions for preparing such foods orbeverages. The methods may comprise the steps of harvesting the plant orplant part, separating grain from other plant parts, crushing,extracting, milling, cooking, canning, packaging or other processingsteps known in the art. The methods or recipes or instructions mayinclude the steps of processing the plant product of the inventionand/or admixing it with other food ingredients, such as heating orbaking the mixture or the product to, for example, at least 100° C. Themethod may include the step of packaging the product so that it is readyfor sale.

In some preferred but not essential embodiments the invention isdirected to products and compositions for use in the herein describedmethods and does not extend to methods for the treatment of the human oranimal body by surgery or therapy and disagnostic methods practiced onthe human or animal body.

In some embodiments the invention is directed to the use of the subjectproducts or compositions in the manufacture of a medicament for interalia increasing intestinal health or, amealiorating one or more symptomsof a condition associated with low levels of dietary fructan, such asdiabetes, obesity, heart disease, hypertension, constipation,osteoporesis and cancer.

Industrial Use

The plant products, preferably grain, may be used in production ofindustrial products such as, for example, ethanol.

The present invention is further described by the following non-limitingExamples.

EXAMPLES Example 1 Illustrative Methods and Materials

Plant Material

The mutant barley (Hordeum vulgare) lines M292 and M342, which werehomozygous for a null mutation in the gene encoding SSIIa, were obtainedfollowing mutagenesis of grains of the barley variety ‘Himalaya’ withsodium azide (Morell et al. 2003 (supra)). Mutant seeds were initiallyselected from progeny grain of the mutagenised population on the basisof a shrunken grain phenotype. This phenotype can be scored readily in alarge population of mutagenized seed. The mutant lines were furthercharacterised by their altered starch properties, reduced SSIIa proteinlevel and activity, and genetically by the presence of a premature stopcodon in the protein coding region of the gene encoding SSIIa (Morell etal. 2003 (supra)).

Both wild-type Himalaya and the mutant plants were grown in a controlledgrowth cabinet at day and night temperatures of 18° C. and 12° C.respectively with a 12 hour day-length. Barley spikes were labelled asat anthesis 2 days after the awns first appeared through the top of theflag leaf containing the enclosed spike. Developing seeds were harvestedat 20 days post anthesis (DPA) and after removal of the embryo thedeveloping endosperm was extruded through the cut surface and stored at−80° C.

Cereal cultivars and other varieties as described herein were obtainedcommercially or from the Australian Winter Cereals Collection, Tamworth,NSW, Australia. Crossing of plants such as barley plants was carried outin the greenhouse by standard methods.

Grain Characteristics

Grain was harvested from plants at maturity. Average seed weight wasdetermined by weighing 100 seeds and expressed as an average weight pergrain (mg). Seed moisture content of grain was measured by standardnuclear magnetic resonance (NMR) methods using an Oxford 4000 NMR Magnet(Oxford analytical instruments Limited). Grain texture was measuredusing the Single-Kernel Characterization system 4100 (Perten InstrumentsInc. Springfield, Ill. 62707 USA) using the RACI Cereal ChemistryOfficial testing method 12-01.

Milling of Grain

Grain was ground to wholemeal that would pass through a 0.5 mm sieve,using a cyclonic mill (Cyclotec 1093, Tecator, Sweden). The wholemealwas then used for the analysis below.

β-Glucan Analysis

α-glucan content was assayed as described in Megazyme Method(AACC32.23), using 20 mg of wholemeal for each of three replicatesamples.

Total Starch Analysis

Total starch content of grain was assayed as described in MegazymeMethod (AACC76.13) using 20 mg of wholemeal for each of three replicatesamples.

Wholemeal was obtained by milling grain. Starch was isolated from thewholemeal using the method of Schulman and Kammiovirta, 1991 (supra).

Analysis of Starch Composition and Characteristics

Amylose and amylopectin contents in the starch of the grain, or theratio of amylose to amylopectin, was determined by Sepharose CL-2B gelfiltration as follows (Gel filtration method). Approximately 10 mg oftotal starch was dissolved in 3.0 ml of 1M NaOH and fractionated on thebasis of molecular weight by chromatography on a Sepharose CL-2B column(Regina et al., 2006 (supra)). The amount of starch in each of thefractions from the column were measured using the Starch Assay Kit(Sigma) according to the suppliers instructions. The total amount ofamylopectin (first peak, higher molecular weight) and amylose (secondpeak, lower molecular weight) was calculated and the ratio or contentsdetermined.

The distribution of chain lengths in the amylopectin of the starch maybe analysed by fluorophore assisted carbohydrate electrophoresis (FACE)using a capillary electrophoresis unit according to Morell et al.,Electrophoresis, 19: 2603-2611, 1998, after debranching of the starchsamples. For example, amylopectin chain length distribution may bemeasured using a P/ACE 5510 capillary electrophoresis system (BeckmanCoulter, NSW Australia) with argon laser-induced fluorescence (LIF)detection. Molar difference plots may be generated by subtracting thenormalized chain length distribution for modified starch from thenormalized distribution for starch from an isogenic non modifiedcontrol.

The gelatinisation temperature profiles of starch samples may bemeasured using a Pyris 1 differential scanning calorimeter (PerkinElmer, Norwalk Conn., USA). The viscosity of starch solutions may bemeasured on a Rapid-Visco-Analyser (RVA, Newport Scientific Pty Ltd,Warriewood, Sydney), using conditions as reported by Batey et al., J.Sci. Food Agric. 74: 503-508, 1997. The parameters that may be measuredinclude peak viscosity (the maximum hot paste viscosity), holdingstrength, final viscosity and pasting temperature. Pasting propertiesmay be measured using the Rapid Visco Analyser as follows. Starch (3.0g) is added to distilled water (25.0 ml) in the DSC pan and the RVA runprofile is: 2 mins at 50° C., heat for 6 mins to 95° C., hold at 95° C.for 4 mins, cool for 4 mins to 50° C., hold at 50° C. for 4 mins. Themeasured parameters are: Peak viscosity at 95° C., Holding strength atend of 95° C. holding period, Breakdown=Peak Viscosity−Holding strength,Final viscosity at end of 50° C. holding period, Setback=FinalViscosity−Holding strength. The software Thermocline for Windows version2.2 (Newport Scientific Pty Ltd, NSW Australia) may be used forcollection and analysis of data.

The swelling volume of flour or starch may be determined according tothe method of Konik-Rose et al. Starch/die Stärke 53:14-20, 2001. Theuptake of water is measured by weighing the sample prior to and aftermixing the flour or starch sample in water at defined temperatures (forexample, 90° C.) and following collection of the gelatinized material.

Lipid Analysis

Total lipid content was assayed by NMR using an Oxford 4000 NMR Magnet,Oxford Analytical Instruments Limited, UK. For each sample, 1 g of seedswas dried at 38.8° C. for 64 hours. The dried seeds were then measuredusing NMR and compared against a pure barley oil controls extracted fromcv. Himalaya or M292 grain.

Protein Analysis

Protein content was estimated by determining the total nitrogen contentof the seed using the method of Dumas (RACI Method 02-03, 2003) andexpressing the result as “protein” by multiplying the value obtained bya factor of 5.7. For each sample 10 mg of wholemeal was used and thenitrogen content was detected by mass spectrometry.

Total Dietary Fibre Assay

The gravimetric method of Prosky et al., J Assoc Off Agric Chem 68: 677,1985 was used to determine total dietary fibre (TDF) of the wholemeal.Duplicate samples were assayed.

Non Starch Polysaccharide Assay

Total neutral non-starch polysaccharides (NSP) were measured by amodification of the gas chromatographic procedure of Theander et al., JAOAC Int 78: 1030-1044, 1995. The modification involved a 2-hourhydrolysis with 1 M sulphuric acid followed by centrifugation to removeinsoluble NSP and a further hydrolysis of the supernatant using 2 Mtrifluoroacetic acid for soluble NSP.

Resistant Starch Assay

An in vitro procedure was used to determine resistant starch (RS)content. The method has two sections: firstly, starch in each sample washydrolysed under simulated physiological conditions; secondly,by-products were removed through washing and the residual starchdetermined after homogenization and drying of the sample. Starchquantitated at the end of the digestion treatment represented theresistant starch content of the sample. Typically, triplicate samples ofwhole meal along with appropriate standards were mixed with artificialsaliva and the resultant bolus incubated with pancreatic and gastricenzymes at physiological pH and temperature. The amount of residualstarch in the digesta was determined using conventional enzymatictechniques and spectrophotometry and the resistant starch content of thesample expressed as a percentage of sample weight or total starchcontent.

On day 1, an amount of sample representing up to 500 mg of carbohydratewas weighed into a 125 mL Erlenmeyer flask. A carbonate buffer wasprepared by dissolving 121 mg of NaHCO₃ and 157 mg of KCl inapproximately 90 mL purified water, adding 159 μL of 1 M CaCl₂.6H₂Osolution and 41 μL of 0.49 M MgCl₂.6H₂O, adjusting the pH to 7 to 7.1with 0.32 M HCl, and adjusting the volume to 100 mL. This buffer wasstored at 4° C. for up to five days. An artificial saliva solutioncontaining 250 units of α-amylase (Sigma A-3176 Type VI-B from porcinepancreas) per mL of the carbonate buffer was prepared. An amount of theartificial saliva solution, approximately equal to the weight of food,was added to the flask. About 15-20 sec after adding the saliva, 5 mL ofpepsin solution in HCl (1 mg/mL pepsin (Sigma) in 0.02 M HCl, pH 2.0,made up on day of use) was added to each flask. The mixing of theamylase and then pepsin mimicked a human chewing the sample beforeswallowing it. The mixture was incubated at 37° C. for 30 min withshaking at 85 rpm. The mixture was then neutralised with 5 mL of 0.02MNaOH. 25 mL of acetate buffer (0.2 M, pH 6) and 5 mL of pancreatinenzyme mixture containing 2 mg/mL pancreatin (Sigma, porcine pancreas at4×USP activity) and 28 U of amyloglucosidase (AMG, Sigma) fromAspergillus niger in acetate buffer, pH6, were added per flask. Eachflask was capped with aluminium foil and incubated at 37° C. for 16hours in a reciprocating water bath set to 85 rpm.

On day 2, the contents of each flask was transferred quantitatively to a50 mL polypropylene tube and centrifuged at 2000×g for 10 min at roomtemperature. The supernatants were discarded and each pellet washedthree times with 20 mL of water, gently vortexing the tube with eachwash to break up the pellet, followed by centrifugation. 50 uL of thelast water wash was tested with Glucose Trinder reagent for the absenceof free glucose. Each pellet was then resuspended in approximately 6 mLof purified water and homogenised three times for 10 seconds using anUltra Turrax TP18/10 with an S25N-8G dispersing tool. The contents werequantitatively transferred to a 25 mL volumetric flask and made tovolume. The contents were mixed thoroughly and returned to thepolypropylene tube. A 5 mL sample of each suspension was transferred toa 25 mL culture tube and immediately shell frozen in liquid nitrogen andfreeze dried.

On day 3, total starch in each sample was measured using reagentssupplied in the Megazyme Total Starch Procedure kit. Starch standards(Regular Maize Starch, Sigma S-5296) and an assay reagent blank wereprepared. Samples, controls and reagent blanks were wet with 0.4 mL of80% ethanol to aid dispersion, followed by vortexing. Immediately, 2 mLof DMSO was added and solutions mixed by vortexing. The tubes wereplaced in a boiling water bath for 5 min, and 3 mL of thermostableα-amylase (100 U/ml) in MOPS buffer (pH 7, containing 5 mM CaCl₂ and0.02% sodium azide added immediately. Solutions were incubated in theboiling water bath for a further 12 min, with vortex mixing at 3 minintervals. Tubes were then placed in a 50° C. water bath and 4 mL ofsodium acetate buffer (200 mM, pH 4.5, containing 0.02% sodium azide)and 0.1 mL of amyloglucosidase at 300 U/ml added. The mixtures wereincubated at 50° C. for 30 min with gentle mixing at 10 min intervals.The volumes were made up to 25 mL in a volumetric flask and mixed well.Aliquots were centrifuged at 2000×g for 10 min. The amount of glucose in50 μL of supernatant was determined with 1.0 mL of Glucose Trinderreagent and measuring the absorbance at 505 nm after incubation of thetubes at room temperature in the dark for a minimum of 18 min and amaximum of 45 min.

Quantification of Sucrose, Hexoses and Fructo-Oligosaccharides

Total sugars were extracted from wholemeal following the method of Lunnand Hatch, Planta 197: 385-391, 1995 with the following modification.Wholemeal such as barley wholemeal (100 mg) was extracted 3 times with10 ml of 80% ethanol (v/v) in a boiling water bath for 10 minutes. Thesupernatant from each extraction was pooled and freeze dried, thenre-suspended in 2 ml milliQ water. The quantities of sucrose, glucose,and fructose were measured using a colorimetric microtiter plateenzymatic assay as described (Campbell et al., Journal of the science offood and agriculture 79: 232-236, 1999; Ruuska et al., Functional PlantBiology 33: 799-809, 2006). Sugars and fructo-oligosaccharides were alsoanalysed by high performance anion exchange chromatography (HPAEC) asdescribed in Ruuska et al., 2006 (supra); both methods resulted incomparable values.

To determine maltose levels, total sugars extracted from barley wholemeal were assayed essentially as described by Bernfeld, In: Colowick S,Kaplan N (eds), Methods in enzymology. Academic, NY, p 149, 1955, usingmaltose standard solutions for comparison, as follows. Total sugars werediluted 10 to 100-fold. Maltose standards (10 tubes) were prepared as0.3 to 5 micromoles per ml. One ml of each dilution of maltose (in totalsugars or maltose dilutions) was mixed with 1 ml of dinitrosalicylicacid colour reagent. The sugar solution was then incubated at 100° C.for 5 minutes and cooled to room temperature. Ten ml reagent grade waterwas added to each tube and mixed well. The samples were measured at A₅₄₀with a spectrophotometer. Maltose was also determined by HPAEC asdescribed above.

Enzyme Assays

Total starch synthase activity in samples such as developing endospermof cereals may be measured by extraction of proteins and assay by themethods described in Libessart et al. Plant Cell 7(8): 1117-1127, 1995or Cao et al., Plant Physiol. 120: 205-215, 1999. The assays use ¹⁴Clabeled ADPG substrate and measure incorporation of the monomer intostarch polymers. Individual isoforms of starch synthase in extracts maybe separated by gel electrophoresis and assayed in-gel (zymogram) asfollows. Extracts from samples such as developing seeds may be preparedusing 50 mM potassium phosphate buffer, pH7.5, 5 mM EDTA, 20% glycerol,10 μM Pefabloc and 0.05 mM dithiothreitol (DTT). After grinding theseeds to a pulp in the buffer or homogenizing the sample, the mixture iscentrifuged at 14,000 g for 15 min at 4° C. and the supernatant drawnoff. The protein concentration in the supernatant may be measured usingCoomassie Protein Reagent or other standard means. Extracts may bestored at −80° C. if the protein extracts are to be run on native gels.For denaturing gel electrophoresis, 100 μl of extract is mixed with SDSand β-mercaptoethanol and the mixtures are incubated in boiling waterfor 4 min to denature the proteins. Electrophoresis is carried out instandard denaturing polyacrylamide gels using 8% polyacrylamideseparating gels overlaid with 4.5% polyacrylamide stacking gels. Afterelectophoresis, the proteins may be renatured by soaking the gels in 40mM Tris-HCl buffers for a minimum of 2 hr, changing the buffer every 30min and using at least 100 mL of buffer for each buffer change. Fornon-denaturing gels, the denaturing step with SDS and β-mercaptoethanolis omitted and SDS omitted from the gels. A starch synthase assay bufferincluding Tris-glycine (25 mM Tris, 0.19M glycine), 0.133M ammoniumsulphate, 10 mM MgCl₂, 670 μg/mL BSA and 1 mM ADPG substrate may be usedto detect starch synthase bands, followed by staining with 2% KI, 0.2%I₂ iodine solution to detect the starch product.

Alternatively, starch synthase or other starch biosynthetic enzymes maybe detected in samples using specific antibodies (ELISA).

cDNA Array

The New South Wales Centre for Agricultural Genomics (NSWCAG) arraycontained 19,635 wheat cDNA clones and 1,613 barley cDNA clones of whichabout 16,000 were unique in nucleotide sequence, wherein a “unique”sequence is defined herein as having less than 80% sequence identity atthe nucleotide level to all of the other sequences. The design of thisarray and the cDNA libraries that contributed to its construction aredetailed in Clarke and Rahman, Theoretical and Applied Genetics, 110:1259-1267, 2005.

Extraction of RNA

To extract total RNA from developing endosperm, samples of eightendosperms each were frozen in liquid N₂ and ground to a fine powderwith the aid of acid washed sand in a mortar containing 2 ml of NTES(0.1 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% (w/v) SDS). Two mlof phenol/chloroform was added to the homogenized mixture and againground well. This mixture was transferred to a capped tube, vortexed forone minute and incubated on ice for 30 minutes. The two phases wereseparated by centrifuging the tubes at 5,500 g for 15 minutes at 4° C.,the aqueous phase transferred to a new tube and an equal volume of 4 MLiCl/10 mM EDTA added to precipitate the RNA. After thorough mixing, thesamples were kept at −20° C. over night. Samples were thawed and spun at7,000 g for 45 minutes at 4° C. The supernatant was discarded and thepellet containing the RNA rinsed with 1 ml of 70% ethanol, dried andre-suspended in 250 μl of water. The soluble fraction was transferred toa new microcentrifuge tube. To remove starch contamination from theisolated RNA, 3.5 μl of 3 M NaOAc and 125 μl of ethanol (EtOH) was addedfollowed by mixing and centrifugation at 13,000 rpm for 10 min at 4° C.The supernatant was removed to a new tube and 21.5 μl of 3 M NaOAc and375 μl of EtOH added to precipitate the RNA. The precipitated RNA wasrinsed with 70% EtOH then dried and re-suspended in 30 μl of water. Todetermine the concentration and purity of the RNA, aliquots were diluted1 in 300 in sterile distilled water and the absorbance measured at A₂₆₀and A₂₈₀.

Microarray Analysis

For microarray analysis, four biological replicates were used for themRNA comparisons. For each labeling experiment, 50 μg of total RNA wasused for both the Cy3 and Cy5 dyes (Amersham Pharmacia, UK), followingthe two-step labelling method of Schenk et al., Proc. Natl. Acad. Sci.U.S.A. 97: 11655-11660, 2000. RNA from the control plant, ‘Himalaya’ waslabelled with Cy3 in the un-swapped replicates. The dye labelling of thesamples was reversed for two of the replicates to minimize any bias incDNA incorporation and photo-bleaching of the fluorescent dyes. Thepre-hybridization of the microarray slides, hybridization of samples andsubsequent washing of the slides to remove unbound target was performedas per the supplied protocol for the CMT-GAPS™ coated microscope slides(Corning USA). The slides were scanned with a GenePix 4000A microarrayscanner (Axon Instruments, Union, Calif., USA). The features wereanalyzed using the GenePix Pro 4 software and unsatisfactorily segmentedfeatures were either manually adjusted or discarded to ensure theintegrity of the data obtained.

The analysis of the microarray data files was carried out usingfunctions contained in tRMA (tools for R Microarray Analysis; Wilson etal., Bioinformatics 19: 1325-1332, 2003) these functions operate as partof a statistical software package called R (http://www.r-project.org/).A detailed description of tRMA is available online(www.csiro.au/gena/trma). The data sets generated from the GenePixsoftware were loaded into tRMA using the “LoadGenePixFile” function.Normalization of log₂ ratio values was performed using the“SpatiallyNormalize” function. This method of normalization correctedfor spatial and intensity-dependent effects of fluorescence across themicroarray slide (Wilson et al., 2003 (supra)). In addition, thepossible biases in fluorescence due to differences in the efficiency ofincorporation of the two dyes and unequal loading of cDNA samples werealso corrected. Using the median values of the normalized log ratios foreach gene in each replicate, differentially expressed genes weredetermined using the “FindDiffExpGenes” function at a stringency levelof 1e−10. Differentially expressed genes are selected as outliers in aGaussian distribution of the entire data set. Therefore, a ratio cut-offwas empirically computed from the normalized log₂ ratio data, which wererescaled and centered in order to make direct comparisons between slidesin all four replicates. The same slide data was also compared using adifferent function. In this analysis after slide normalization the“FindDiffExpGenes” function was performed on the individual slides atthe default stringency (1e−3). Then “CompareInterestingGenes” functionwas used to find the differentially expressed genes common to allreplicates.

To obtain the nucleotide sequence of cloned genes or, for example, toconfirm the identity of the differentially expressed genes, the cloneswere sequenced using 0.12 μg of PCR amplified insert as the template,from both the 3′ and 5′ ends using primers such as M13/pUC reverse andforward primers and Big Dye Terminator Cycle Sequencing (ABI).

RNA Electrophoresis and Hybridization Conditions

For each sample, 10 μg of total RNA was separated in a 1.4%agarose-formaldehyde gel (w/v) and transferred to Hybond N+ membrane(Amersham Pharmacia Biotech UK Ltd.) using the standard alkali transferprotocol supplied by the manufacturer.

Probes used for RNA hybridizations were made by amplifying the insertsfrom cDNA clones. PCR SuperMix (Invitrogen) was used with 3 ρmoles ofeach primer and 50 ng template DNA in 10 α1 reaction mix. The probeswere amplified under the following conditions; cycle 1, 94° C. for 5minutes; cycle 2, 94° C. for 30 seconds, 55° C. for 30 seconds and 72°C. for 2 minutes, repeat cycle 2 for 35 cycles. The inserts were labeledusing the “Megaprime™ DNA labelling system” (Amersham Biosciences). Inaddition the clone pTa250.2, containing the coding sequences of theribosomal genes (Appels and Dvorak, Theoretical and Applied Genetics,63: 337-348, 1982) was used to estimate the uniformity of loading forthe RNA from Himalaya and M292 onto the gels.

Hybridization of probes was at 65° C. in Khandjian hybridization buffer(Khandjian, Bio/Technology, 5: 165-167, 1987). The membrane was washedonce for 30 minutes at 65° C. with 2×SSC and 0.1% (w/v) SDS, and twicefor 40 minutes at 65° C. with 0.2×SSC and 0.1% (w/v) SDS, whichcorresponds to a high stringency wash. The membrane was exposed using aFujifilm FLA-5000 series fluorescent image analyzer system and the imageobtained was analyzed using the Multi Gauge version-2 analysis software(Fuji Photo Film Co. Ltd., 26-30 Nishiazabu, 2-Chome Minato-ku Tokyo106-8620, Japan). The variation in hybridization intensities between theHimalaya and M292 RNA samples was measured (pixels per mm²) and acorrection was made for the level of background hybridization to the gellanes.

Example 2 Composition and Functional Parameters of M292 Barley Grain

A detailed analysis was undertaken of the composition of mature grainfrom the barley SSIIa mutant M292 with a comparison to wild-type grain(Himalaya) grown under the same conditions. The results are summarizedin Table 1.

M292 producing thinner grains with an unfilled central region(“shrunken”). The moisture content of the M292 grain was 10.2% asmeasured by the NMR method compared to 10.6% for Himalaya. The averagegrain weight for M292 was 36.4 mg/grain compared to Himalaya at 46.3mg/grain, which represents a 21% reduction for M292, primarily due toreduction in the starch content from 27.7 mg/grain to 10.6 mg/grain (62%reduction). The reduction in starch came mostly from less amylopectin(84% reduction) while the amylose level was reduced by only 25%.Consequently, the percentage starch as amylose increased from about 37%in the wild-type to about 73% in M292 as determined by the gelfiltration method.

TABLE 1 Grain composition of barley M292 at maturity Ratio Content inHimalaya Content in M292 M292/Him. (mg/ (% of total (mg/ (% of total (ona mg per grain) grain) grain) grain) grain basis) Seed weight 46.3 36.40.79 Starch 27.7 59.9 10.6 29.1 0.38 amylose 10.4 26.1 7.8 21.4 0.75amylopectin 17.3 43.6 2.8 7.7 0.16 Protein 5.9 12.8 7.0 19.3 1.2 TotalNSP 5.6 12.1 7.4 20.3 1.3 Lipid 1.5 3.2 2.5 6.9 1.7 Sugars (total) 0.070.16 0.6 1.6 8.6 glucose 0.005 0.08 16.0 fructose 0.005 0.09 18.0sucrose 0.060 0.39 6.5 maltose 0.005 0.03 6.0 Fructan 0.1 0.2 4.2 11.542.0 Ash 1.1 2.4 0.8 2.2 0.73 Moisture 4.9 10.6 3.7 10.2 Total content47.4 102.4 36.8 101.1 (incl. water) Resistant starch 0.3 0.7 1.3 3.6 4.3Beta-glucan 2.7 5.8 3.6 9.9 1.3 Dietary fibre 7.2 15.6 9.2 25.3 1.3Arabinoxylan 2.5 5.5 3.1 8.6 1.2 Note: Total content is a sum ofcomponents in bold type; NSP: Non starch polysaccharide.

The reduced starch amount in M292 was compensated for in part byincreases in the amounts of non-starch components. The contents ofprotein, total NSP and lipid (measured as mg per grain), which togethermake up about 46% of the grain weight of M292, were increased by 1.2,1.3 and 1.7-fold, respectively. As a percentage of total M292 grainweight, the increases were even greater. The levels of free sugars wereincreased in M292 by about 8-fold in total, with individual sugarsincreasing between 6- and 18-fold. Of the other carbohydrate components,α-glucan and arabinoxylans were increased in the mutant by 1.3 and1.2-fold, respectively. Unexpectedly and surprisingly,fructo-oligosaccharides (fructans) were increased massively by42.0-fold. The extent of this increase was most surprising sinceα-glucan and arabinoxylans, which are also polymeric carbohydrates, wereincreased only modestly. The increase for fructan in terms of percentageof total grain weight was from 0.2% for Himalaya to 11.5% for M292. Itis believed such a high level of fructan has never before been seen in agrain.

The total carbohydrate content (including starch, total NSP, free sugarand fructan) was reduced from 33.5 mg/grain present in wild-typeHimalaya grain to 22.8 mg/grain in M292. The amount of resistant starch(RS) as determined by the in vitro method as described in Example 1 wasincreased in M292 by 4.3 fold (mg per grain). This indicated a muchincreased extent of protection of the starch from amylase digestion,presumably due to an altered starch granule structure and related to theincreased proportion of amylose in the starch. Increased levels oflipid, α-glucan and fructan are also thought to have contributed to theincreased level of RS.

The composition of the soluble carbohydrate components was furtherexamined by HPAEC to study the changes in the oligosaccharides. Thechromatography profiles (FIG. 1) confirmed the increased content ofsucrose, maltose and hexoses in M292, as well as increased levels of arange of fructo-oligosaccharides, which showed a degree ofpolymerization (DP) of from 3 up to about 12. The wild-type Himalayagrain contained negligible or undetectable levels offructo-oligosaccharides having a DP of about 6 or above.

Changes in cell wall polysaccharides can affect the hardness of themature barley grain (Tsuchiya et al., Physiologia Plantarum 125:181-191, 2005; Fincher and Stone, Advances in Cereal Science and Technol8: 207-295, 1986). As there were significant changes of total NSP inM292, grain hardness was measured to determine the level of changes. Themeasurements were taken using the Single-Kernel Characterization System(SKCS) to obtain an average hardness index (HI) based on measurementsfrom 300 grains. For M292 the HI value was 109±15 and the HI value forHimalaya was 84±17, indicating an increase in grain hardness for themutant.

The contents of protein, total NSP and lipid (measured as mg per grain)which together make up about half the grain weight of M292, wereincreased in absolute amount and as a percentage of total grain weight,by a 1.2, 1.3 and 1.7-fold change respectively. Of the carbohydratecomponents which may be particularly significant in a nutritionalcontext, α-glucan, arabinoxylans and fructo-oligosaccharides, wereincreased in the mutant by, 1.3-, 1.2-, and 42.0-fold, respectively. Theresistant starch (the proportion of the total starch that is resistantto digestion in the human small intestine) was also increased in M292 by4.3 fold change when expressed as mg per grain. The total content ofcarbohydrates (including starch, total NSP, free sugar and fructan) inM292 reduced to 22.8 mg compared to 33.5 mg present in wild-typeHimalaya on a mg per grain basis.

Discussion

The identification and initial characterization of the barley M292mutant was described by Morell et al. 2003 (supra) and Topping et al.,2003 (supra). Using linkage analysis and subsequent sequencing of thecandidate gene, Morell et al. 2003 (supra) demonstrated that themutation was caused by a stop codon introduced into SSIIa and resultedin a shrunken grain in which the seed weight was reduced from an averageof 46 mg in Himalaya to 36 mg in M292 (Table 1) producing a thinner seedwith an unfilled central region. The decreased starch, grain weight andmodified starch composition observed here were consistent with previousstudies (Morell et al. 2003 (supra)). The inventors have also shownincreased levels of protein, total NSP, and lipid both on an individualgrain basis and as a percentage, consistent with determinations reportedearlier (percentage of total grain basis, Bird et al., 2004a (supra)).

In addition, the inventors have also shown, for the first, timesignificantly increased levels of free sugars andfructo-oligosaccharides. As well as increased resistant starch,β-glucan, and dietary fibre, the higher fructo-oligosaccharidesdetermined here are expected to be important for providing beneficialdietary outcomes.

Example 3 Identification of Differentially Expressed Genes Using a cDNAMicroarray

A microarray containing 19,635 wheat clones and 1,613 barley clones ofwhich about 16,000 were unique sequences was obtained from the New SouthWales Centre for Agricultural Genomics (NSWCAG). A “unique” sequence isdefined herein as a sequence having less than 80% sequence identity atthe nucleotide level to all other sequences in the set. The design ofthis array and the cDNA libraries that contributed to its constructionare detailed in Clarke and Rahman, 2005 (supra).

The array was hybridized using total RNA from developing endosperm (20DPA) of M292 or the wild type ‘Himalaya’ (control). At this time point,the levels of starch and protein were increasing in the developingbarley grain. It was expected that the transcriptional changes to thestarch biosynthetic pathway, caused by the mutation in the SSIIa gene,would be most evident at this timepoint. Seeds at the same age andmorphological stage of development were selected from a single spike torepresent a sample. The seeds collected from a different spikerepresented a replicate biological sample.

Four biological replicate experiments for M292 and the control Himalayawere used to compare the transcription profiles in developing endospermat the mid grain fill stage. Differentially expressed genes wereidentified from the median data sets of differentially expressed genesfrom the four microarray experiments. The stringency level used toselect the genes was 1e−10. This level of stringency was determinedempirically to give the least number of false positive results. From thetRMA analysis, 42 array features were identified as differentiallyexpressed, 20 of which were up-regulated and 22 down regulated in M292compared to Himalaya. All of the clones identified were verified bysequence analysis from both the 3 prime and 5 prime ends to ensure thatthe annotation of the clone was correct. These results showed that oneclone was a chimera and this clone was not analyzed further. Using thesequence data, a sequence homology search was made against the TIGR(http://www.tigr.org) tentative consensus sequence (TC) data base usingeither the barley or wheat Gene Index, depending on the origin of theclone on the microarray. From this comparison the 41 cDNA clones couldbe grouped into 23 different genes. A verification of the expressionchanges was then undertaken by RNA blot-hybridization using one clone asa representative for each TC. The expression changes were verified usingan RNA gel blot analysis in which RNA was isolated from a fifthreplicate endosperm sample. The change in expression for 6 clones wasnot confirmed and these clones were not analyzed further.

The 17 clones which passed the quality control conditions, their TCsequence and the expression change, relative to Himalaya, for both themicroarray and RNA gel blot analysis are listed in Table 2. These geneshave been grouped into four categories, being carbohydrate related,defense, stress response and those genes for which a biological functionhas not yet been established. In Table 2, column 4 presents themicroarray results (M292/Himalaya) and column 5 presents the RNA gelblot analysis results. Therefore, the numbers in these columns representthe fold change in gene expression in M292 above or below the Himalayacontrol. Clone pTa250 (Appels and Dvorak, 1982 (supra)) was used as acontrol sequence for the RNA blot hybridizations.

TABLE 2 Differentially expressed genes in M292 developing grain TIGRGenBank Micro- TC ID Name Array Northern Carbohydrate related genes250803 AL812383 β-D-glucan exohydrolase 2.33 4.60 250388 BQ607866sucrose synthase 2.47 1.25 139354 CV054497 serpin 0.30 0.10 249933BQ609223 β-amylase 0.27 0.20 269042 BQ606784 starch synthase 0.17 0.03Defense related gene 146614 CV055257 alpha-amylase inhibitor 0.36 0.53BDAI-I Stress protein related genes 232242 BG263730 t.complex protein2.56 2.10 246814 AL818443 dnaK-type protein 2.30 1.60 250385 BG314518heat shock protein 80 2.27 3.22 264292 BE442600 heat shock protein 702.25 2.00 139615 CV056993 prohibitin 2.04 1.70 Genes of unassignedfunction 246973 BQ606826 annexin p33 2.85 4.10 232395 X83881S-adenosylmethionine 2.37 1.30 decarboxylase 233204 BE444846 Rubberelongation factor 2.14 3.32 249807 BQ608029 O.s r40g2 protein 2.26 10.00234638 BQ606719 Puro/hordoindoline-a 0.40 0.58 147907 CV060362 No Hiteither blast x or n 0.32 0.27

Interestingly, many genes known to play a role in starch biosynthesisthat were represented on the microarray did not show differentialexpression in M292 compared to Himalaya. These were as follows (withGenbank Accession Nos.): ADP Glucose pyrophosphorylase small subunit(AL815034); ADP Glucose pyrophosphorylase large subunit (AL814437);Granule bound starch synthase I (BQ608470); Granule bound starchsynthase II (BE497955); Starch synthase I (AL815975); Starch synthaseIII (AL811419); Starch branching enzyme I (AL816520); Starch branchingenzyme IIa (AL812818); Starch branching enzyme IIb (BF201559);Isoamylase: glycogen 6-glucanohydrolase (BE422551); Alpha amylase(AL809888).

At 20 DPA in barley grain, 4 differentially expressed genes other thanSSIIa were identified that have been related to the storage of carbonthrough sucrose uptake in the endosperm. Of these 4 genes, 2 were upregulated in M292 (β-D-glucan exohydrolase and sucrose synthase) and 2down regulated (serpin, β-amylase and ssIIa).

The SSIIa transcript was almost undetectable by RNA gel blot analysis,being reduced to only 3% of the wild-type level. This result wasconsistent with the absence of SSIIa proteins in the M292 developingendosperm and mature grain as observed by Morell et al. 2003 (supra).The reduction by 97% may be a consequence of rapid turnover of the mRNAby the nonsense-mediated mRNA decay pathway. The smaller reduction ofssIIa transcript observed in the microarray experiment compared to theRNA gel blot hybridization result, may have been due to some crosshybridization of transcripts from other members of the starch synthasefamilies. This example shows the importance of confirming resultsobtained from the arrays with RNA gel blot hybridization or RT-PCRexperiments, as these methods can be targeted to specific members of thegene families.

The function of β-amylase in the germinating grain is to removesuccessive maltose units from the non reducing ends of the starch chainto provide a source of carbon, but it is also synthesized andaccumulates during grain development (MacGregor et al., CerealChemistry, 48: 255-269, 1971). Guerin et al., Journal of Cereal Science,15: 5-14, 1992 showed that β-amylase was present in the seed as a boundinactive complex with Protein Z (serpin) that is associated with thestarch granules. In this study the transcript levels of both serpin(protein Z type) and β-amylase were reduced in the mutant lineindicating that these proteins may be co-regulated in the developinggrain.

The expression of sucrose synthase (SuS) was slightly up-regulated inM292 endosperm. The catalysis of sucrose and UDP to form UDP-glucose andfructose is carried out by SuS in a reversible reaction. The UDP-glucoseproduced by SuS can be converted via the combined action of UDP-glucosepyrophosphorylase and phosphoglucomutase to give Gluc-1-P and Gluc-6-Pwhich can enter glycolysis or be used in starch synthesis (Winter andHuber, Critical Reviews in Plant Sciences 19: 31-67, 2000).Alternatively, it can provide the substrate for the synthesis ofcellulose, callose, or other cell wall polysaccharides. It appears thatthe mutation ssIIa in M292 disrupted the utilization of sucrose forstarch synthesis, associated with an increase in the level of sucrose(6.5 fold increased in M292, Table 1) and increased SuS expression.

Other sucrose regulated, or UDP-glucose utilizing enzymes were notdifferentially expressed in this analysis. For example, there were 11clones present on the cDNA array relating to a range of cellulosesynthase or cellulose synthase-like genes and 3 callose synthase genes,none of which were differentially expressed. However, there was asignificant increase (33% on a mg per grain basis) of β-glucan contentin the M292 grain and UDP-glucose is a substrate for the synthesis ofthis polysaccharide. While the level of fructo-oligosaccharides weregreater in mature M292 grain, than in Himalaya, genes encoding enzymesof fructan biosynthesis, such as sucrose:sucrose 1-fructosyltransferaseand sucrose:fructan 6 fructosyltransferase, were not differentiallyexpressed in this comparison.

There was an increase in expression of the gene β-D-glucan exohydrolasein M292. This exohydrolase has a broad range of substrate specificitywhich presents a problem in assigning a specific target molecule forthis enzyme. Hrmova and Fincher, Plant Molecular Biology, 47: 73-91,2001 suggested the β-D-glucan exohydrolases be classified aspolysaccharide exohydrolases because they can hydrolyze a range ofpolysaccharides and oligosaccharides. This enzyme is usually found insituations where cell wall degradation or modification is occurring(Harvey et al., Physiologia Plantarum 113: 108-120, 2001; Hrmova andFincher, 2001 (supra)). A possible role for this enzyme in M292 is thedegradation of those endosperm cells that are not filled in the shrunkenmutant grain, or recovering/recycling glucose as proposed by Hrmova andFincher, 2001 (supra).

Previous analysis showed that the quantities of starch branching enzymesIIa, IIb are similar in the wild type and M292 seed (Morell et al. 2003(supra)). The observation that the protein level was unchanged isconsistent with the present transcription analysis where no differentialexpression was observed for branching enzyme IIa or IIb.

The role of the other differentially expressed genes identified in thisanalysis in M292 is unknown.

In summary, in wild-type Himalaya sucrose transported into the grain canbe efficiently converted to storage carbohydrates. Two major forms ofthe carbohydrates in Himalaya are starch (82.8% of total carbohydrate)and NSP (16.7% of total carbohydrate), with free sugar and fructan beingthe minor components. In M292, due to the ssIIa mutation, this processhas been disrupted, leading to the reduction of total starch to 46.6% oftotal carbohydrate. It is proposed that the ssIIa mutation leads to theaccumulation of sucrose that up-regulates the expression of SuS. Thesucrose and UDP-glucose (synthesized by SuS) are then used as thesubstrates for the synthesis of NSP (32.5% of total carbohydrate),fructan (18.4% of total carbohydrate) and free sugar (2.6% of totalcarbohydrate) instead of starch. The differential expression ofUDP-glucose pyrophosphorylase was not detected, although this cDNA wason the microarray.

Example 4 Determination of Fructan Content in Grain

Method for Quantification of Fructo-Oligosaccharides (Fructans)

Total sugars were extracted from wholemeal following the method of Lunnand Hatch, 1995 (supra) with the following modification. Wholemeal isdefined herein as the product obtained by milling mature grain, withoutsubsequent fractionation (e.g. sieving) to remove the bran. Thereforewholemeal contains all of the components in the grain.

The wholemeal was extracted 3 times with 10 ml of 80% ethanol (v/v) in aboiling water bath for 10 minutes. The supernatant from each extractionwas pooled and freeze dried, then re-suspended in 2 ml milliQ water. Thequantities of sucrose, glucose, and fructose were measured using acolorimetric microtiter plate enzymatic assay as previously described(Campbell et al., 1999 (supra); Ruuska et al., 2006 (supra)). Sugars andfructo-oligosaccharides (fructans) were also analysed by highperformance anion exchange chromatography (HPAEC) as described in Ruuskaet al., 2006 (supra). Both methods gave similar results.

Comparison of Fructan Contents Among Barley Varieties

Barley lines with different genetic backgrounds were used in acomparison of fructan contents. The barley lines used were: (1) linesthat contain a starch synthase IIa (SSIIa) mutation (Barley M292, BarleyM342 and Tantangara×292 double haploid (DH)); (2) a barley line with awaxy mutation inactivating GBBSI (Waxiro); (3) a high amylose barley(with 45% amylose, and a mutated gene(s) designated amoI); and (4)wild-type barley lines (Gardiner, Schooner, Himalaya, Sloop, Namoi andGlacier). These lines were grown at Francis, South Australia.Tantangara×292 DH is a barley line from a double haploid population fromthe crossing between Tantangara and Barley M292.

The results are shown in Table 3, where the fructan content representsthe sum amount of glucose and fructose after hydrolysis minus theamounts of free glucose, fructose and sucrose (without hydrolysis). In acompletely unexpected result, the data indicated that the barley linescontaining a mutation in the SSIIa that inactivated SSIIa producedrelatively high levels of fructan (shown in mg/g grain), whereas thebarley lines without the SSIIa mutation produced relatively low levelsof fructan. The observation was even more surprising in view of the lackof any change in the expression levels of two genes involved in fructanbiosynthesis (Example 3).

TABLE 3 Analysis of fructan levels in grain of barley varietiesUn-hydrolysed Glucose Fructose Sucrose Hydrolysed Barley content contentcontent Maltose Glucose Fructose Fructan line (mg hexose equiv/g dryweight) content content content content^(a) Barley M292 2.3 2.4 10.6 0.941.2 75.0 100.1 49.0 97.9 130.7 Gardiner 0.6 0.7 6.5 0.3 15.9 28.1 35.810.6 21.4 23.8 Schooner 0.8 0.8 7.7 0.5 19.0 28.2 37.4 14.3 22.0 26.4Himalaya 0.1 0.1 1.3 0.1 1.3 2.8 2.6 1.2 2.5 2.3 Waxiro 0.7 0.8 7.1 0.615.8 21.8 28.3 11.4 20.4 22.5 Sloop 0.6 0.6 7.0 0.4 10.1 17.4 18.9 10.017.3 18.8 Barley mutant 342 1.4 1.7 10.6 0.9 33.6 66.3 85.3 42.7 93.8121.9 36.3 77.3 99.0 Namoi 0.6 0.6 7.0 0.4 8.7 16.4 16.5 Tantangara 0.50.5 7.6 0.3 10.6 20.1 21.8 Tantangara × 292 1.9 3.2 11.0 1.0 41.1 95.4119.5 DH HA Glacier 0.5 0.6 6.1 0.2 6.9 13.8 13.3 Glacier 0.8 0.7 5.70.5 7.3 14.3 14.0 ^(a)Fructan content expressed as mg/g wholemeal, whichis essentially mg/g grain

The effect on fructan levels of the SSIIa mutation in different geneticbackgrounds was then examined. The SSIIa mutation was transferred bybackcrossing (one cross and three backcrosses, with single seed descent,equivalent to BC3F4) to two different barley varieties, namely cultivarsSloop and Tantangara. Progeny lines 250 to 374 contained the SSIIamutation in a barley Sloop background and lines 703 to 886 had a barleyTantangara background. K4 was a black barley grain with wildtype starch,without the SSIIa mutation, grown in the same field. Total sugars wereextracted from 100 mg dry weight wholemeal as described above in section4.1.

The results are shown in Table 4, which indicated that the SSIIamutation in both the Tantangara and Sloop barley varieties produced ahigh fructan content (shown in mg/g wholemeal).

TABLE 4 Comparison of fructan contents in ten selected breeding linesUn-hydrolysed Glucose Fructose Sucrose Hydrolysed Barley content contentcontent Maltose Glucose Fructose Fructan lines (mg hexose equiv/dryweight) content content content content 250 2.3 2.4 13.4 1.7 20.2 48.548.9 348 2.6 2.9 14.9 1.4 16.7 43.7 38.6 363 2.3 2.8 13.3 1.2 19.9 49.149.4 374 2.5 3.3 13.9 1.6 20.0 52.5 51.2 703 2.2 2.7 12.9 1.1 20.5 54.155.6 871 1.8 2.1 13.5 1.5 13.2 41.3 35.6 926 2.3 3.2 12.7 1.2 20.3 52.653.4 930 2.1 2.9 11.9 1.1 13.9 48.8 44.6 886 1.5 2.3 12.9 1.6 18.6 46.446.6 K4 0.4 0.4 6.7 0.2 4.2 8.8 5.2Analysis of Fructan Content in SSIIa Mutant Wheat Grain

Total sugars were extracted from 100 mg dry weight wholemeal asdescribed above in section 4.1 for a wheat SSIIa triple null mutant(mutant in the A, B and D genomes of wheat) and the correspondingwildtype wheat Sunco. These were analysed for fructan content as for thebarley grain.

The results (Table 5) indicated that the wheat grain of the SSIIa triplenull mutant contained increased fructan levels compared to thecorresponding wild-type wheat (Sunco) grain for all growingenvironments, but to a varying extent. For example, comparison betweengrain of the SSIIa null line B63 and the wild-type line B70 indicated a2-3 fold increase in fructan in the mutant grain. The data in Table 5also indicated that amounts of glucose, fructose, sucrose and maltosewere sometimes increased in the null wheat. The data also showed thatthe wheat SSIIa triple null line had less hexoses than the control lineBW26, but sucrose was not significantly altered. Overall, these resultsindicated that an SSIIa mutation increased fructan content not only inbarley, but also in wheat.

TABLE 5 Analysis of fructan contents in wheat SSIIa mutant grainUn-hydrolysed Glucose Fructose Sucrose Hydrolysed: Barley contentcontent content Maltose Glucose Fructose Fructan lines (mg hexoseequiv/g dry weight) content content content content B29 GES 2003 1.171.06 10.08 1.96 11.35 25.32 22.39 B63 GES 2003 0.58 0.52 13.33 1.6417.99 40.14 42.06 B70 GES 2003 wt^(a) 0.21 0.14 4.34 0.14 5.73 12.4913.39 B23/24 GES 2004 (100) 0.33 0.30 7.84 0.52 11.36 24.21 26.58 B29GES 2004 0.50 0.38 10.19 1.48 9.88 27.63 24.97 B63 GES 2004 0.68 0.7313.10 1.60 19.78 45.88 49.55 A9 GES 2004 wt 0.54 0.53 11.78 0.45 8.4819.05 14.24 A113 GES 2004 wt 0.75 0.73 12.02 1.34 8.54 19.34 13.03 B70GES 2004 wt 0.14 0.06 7.86 0.30 9.49 22.44 23.57 B29 Griffith 2005 1.531.31 11.36 1.78 18.22 38.78 41.01 B70 Griffith 2005 wt 1.75 2.93 2.100.29 6.82 16.56 16.30 B29 GES 2006 2.51 2.36 13.42 2.06 19.40 37.3236.38 B63 GES 2006 1.21 0.98 16.40 1.09 23.21 57.98 61.51 A113 GES 2006wt 0.52 0.45 7.42 1.00 8.52 19.52 18.64 B70 GES 2006 wt 0.20 0.11 4.530.00 10.86 22.66 28.69 ^(a)wt indicates these lines were wild-type forSSIIa

The lines used in this analysis were generated by crossing a parentalSSIIa null wheat plants (SGP-1 null wheat) and wheat plants of cultivarSunco. B29 GES 2003 and B63 GES 2003 were SSIIa mutant in all threegenomes (triple nulls) and were grown at Ginninderra ExperimentalStation in 2003. B70 GES 2003 wt was wildtype for SSIIa and was alsogrown at Ginninderra Experimental Station in 2003. B23/24 GES 2004(100), B29 GES 2004 and B63 GES 2004 were mutant for SSIIa (triple null)and were grown at Ginninderra Experimental Station in 2004. A9 GES 2004wt, A113 GES 2004 wt and B70 GES 2004 wt were wildtype for SSIIa andwere grown at Ginninderra Experimental Station in 2004. B29 Griffith2005 was mutant for SSIIa (triple null) and was grown at Griffith in2005. B70 Griffith 2005 wt was wildtype for SSIIa and was grown atGriffith in 2005. B29 GES 2006 and B63 GES 2006 were SSIIa mutant(triple nulls) and were grown at Ginninderra Experimental Station in2006. A113 GES 2006 wt and B70 GES 2006 wt were wildtype for SSIIa andwere grown at Ginninderra Experimental Station in 2006.

Analysis of Fructan Content in High Amylose Barley Grain

Total sugars were extracted from 100 mg dry weight wholemeal of severalbarley lines transgenic for SBEIIa and/or SBEIIb RNAi constructs, andwild-type barley grain of cultivar Golden Promise. Those containing theSBEIIa RNAi constructs had strongly downregulated expression of theSBEIIa gene and consequently the grain starch in these lines waselevated to about 80% amylose.

The results indicated that the transgenic barley grain containing boththe SBEIIa RNAi and SBEIIb RNAi constructs (transgenic barley #22) hadelevated fructan levels compared to the wildtype barley grain (GoldenPromise), but the transgenic lines containing either of the singleconstructs, transgenic barley #20, containing the SBEIIa RNAi constructand #21, containing the SBEIIb construct, were not elevated for fructanlevels in the grain.

Analysis of Fructan Content in High Amylose Wheat Grain

Total sugars were extracted from 100 mg dry weight wholemeal asdescribed above in section 4.1 for a high amylose wheat line which wastransgenic for an inhibitory SBEIIa RNAi construct and thereforeelevated in amylose content to about 80% amylose (Regina et al., 2006(supra)) and a corresponding wildtype wheat BW26. These were analysedfor fructan levels in the grain as for the barley, above. SBEIIa andSBEIIb refer to starch branching enzymes IIa and IIb, respectively.

The results indicated that high amylose wheat grain containing theSBEIIa construct (#25, #26), a transgenic wheat line containing theSBEIIb construct (#27, not elevated in amylose level) and grain of atransgenic wheat line containing an SSI RNAi construct and havingreduced starch synthase I activity did not have substantially increasedfructan content compared to the wild-type wheat transgenic wheat line(#28).

Effects of Growing Conditions on Fructan Content

Further experiments were carried out to determine the effects of growingconditions on the level of fructan in grain across a range of barleylines containing the SSIIa mutation.

Lines 250 to 374 and lines 703 to 926 were as described above. 2001-292:Barley M292 was grown at Ginninderra Experiment Station (GES), ACT in2001. 2001-292: Barley M292 was grown at Forbes (NSW) in 2001. 2002-292:Barley M292 was grown at Colleambally in 2002. 2003-292: Barley M292 wasgrown at Forbes in 2003. 2003-292: Barley M292 was grown at Colleamballyin 2003.

The data showed that the fructan levels were elevated across a range ofbarley genetic backgrounds each containing the SSIIa mutation, and avariety of growing environments.

TABLE 6 Effects of growing conditions on fructan content Line Fructanlevel Line No Growing site name (mg/g grain) 2003 Black mountainglasshouse, ACT 250 107.1 2003 Black mountain glasshouse, ACT 266 53.02003 Black mountain glasshouse, ACT 348 59.8 2003 Black mountainglasshouse, ACT 363 115.2 2003 Black mountain glasshouse, ACT 374 98.32003 Black mountain glasshouse, ACT 703 174.2 2003 Black mountainglasshouse, ACT 871 99.6 2003 Black mountain glasshouse, ACT 886 126.92003 Black mountain glasshouse, ACT 926 103.9 2003 Black mountainglasshouse, ACT 930 164.0 2001 Ginninderra Experimental Station, ACT 29268.9 2001 Forbes 292 76.6 2002 Colleambally 292 37.0 2003 Forbes 29240.4 2003 Colleambally 292 42.1

Example 5 Large Scale Production of Fructan

Having about 10% fructan, the barley grain mutant in SSIIa can be usedfor the isolation and purification of fructan as well as other productssuch as high amylose starch and β-glucan. Such production from grainwhich can be readily produced in broadacre agriculture will becost-effective relative to existing methods of fructan production, forexample, involving the extraction of inulins from chicory. BarleyMaxgrain contained at least 5% fructan by weight, or when grown under someconditions, at least 10% fructan.

Large scale extraction of fructan can be achieved by milling the grainto wholemeal flour and then extracting the total sugars includingfructans from the flour into water. This may be done at ambienttemperature and the mixture then centrifuged or filtered. Thesupernatant is then heated to about 80° C. and centrifuged to removeproteins, then dried down. Alternatively, the extraction of flour can bedone using 80% ethanol, with subsequent phase separation usingwater/chloroform mixtures, and the aqueous phase containing sugars andfructan dried and redissolved in water. Sucrose in the extract preparedeither way may be removed enzymatically by the addition ofα-glucosidase, and then hexoses (monosaccharides) removed by gelfiltration to produce fructan fractions of various sizes. This wouldproduce a fructan enriched fraction of at least 80% fructan.

Example 6 Production of Food Products

The mature grain as harvested, processed grain, wholemeal or flourobtained from the grain can be used to produce food products forconsumption by humans or other mammals by any method known to persons ofordinary skill in the art. For example, wholemeal bread may be made bysubstituting from 15-30% (w/w) or even more of the wholemeal used inbread recipes with wholemeal from any one of the SSIIa mutant graintypes described herein.

Example 7 Compositions for Treatment

A composition of purified fructan in the form of a capsule for oraltreatment may be prepared by filling a standard two-piece hard gelatincapsule with 500 mg of the agent or compound, in powdered form, 100 mgof lactose, 35 mg of talc and 10 mg of magnesium stearate.

Example 8 In Vitro Fermentation Studies and Rat Feeding Trial

In vitro experiments and subsequent feeding trials in rats examined thefermentative and physiological properties of barley grain mutant 292extracts.

In Vitro Fermentation Studies

An anaerobic static batch culture system was used to model human colonicfermentation. This simulation system is widely used internationally andyields reliable and reproducible results. Inoculum for the system wasfreshly voided faeces sourced from healthy adult subjects consumingtheir habitual diets. After collection, faecal samples were homogenisedand suspended at 10% w/v in sterile anaerobic PBS.

A series of studies employing a completely randomised experimentaldesign was used to investigate the fermentative properties of novelcereal product (extracts of barley grain mutant 292). Predeterminedamounts of these products were added to incubators. Referencecarbohydrate substrates (glucose, lactulose and inulin), at comparablelevels of addition, were included in each assay run. Quadruplicateincubations were performed in an anaerobic chamber for the cereal testproducts, standard substrates as well as for the Control (blank; noexogenous substrate added to the fermentor). Briefly, test products (andstandards) were pre-weighed into sterile screw-capped sterilefermentation vessels and the carbon-limited fermentation mediacomprising carbonate buffer and macro- and micronutrients added to aachieve the predetermined volume and pH (7.0). After a period ofequilibration an aliquot of inoculum was added to each fermentor. Thesewere then capped, sealed and incubated at 37° C. with constant shaking.Incubations containing no added substrate (blank) were included in eachassay run. After 24 hours, the ferments were frozen at −20° C. to awaitbiochemical analysis using standardised procedures. Short chain fattyproduction (SCFA) of the test products and standards from the study areshown in FIG. 2. The results show that at the lower dosage (1%) theextracts of the barley grain mutant 292 produced a comparablefermentation pattern to that of inulin.

Rat Feeding Trial

The rat study was designed to corroborate and extend the findings of thein vitro fermentation experiments. Such techniques are capable ofproviding only preliminary information as they are clearly unable tofully simulate the complex physical, microbial and chemical processesthat occur along the gastrointestinal tract.

The primary aim of the study was to investigate the fermentationproperties of extracts of barley grain mutant 292 stem. These novelproducts were compared against a negative control and an established(commercial) prebiotic.

The study comprised a completely randomised design involving 5 dietarytreatment groups with 10 rats per group. The treatments were:

Negative control (basal diet containing no additional fibre); Barleygrain mutant 292 extract, included at 2% and 5% (by weight) of the diet.

Positive controls: commercial oligofructose at 2% and 5% of the diet.

The major experimental endpoints were cecal SCFA concentrations andpools.

Briefly, rats (approximately 200 g live weight) were acclimatised for 7days before being assigned randomly to treatment groups. A non-purifiedcommercial chow was fed during the adaptation phase and treatment dietswere fed for the subsequent 2 weeks of study. Rats were maintained inwire-based cages except for the final 4 days of study when they werekept in metabolic cages to enable record daily feed and water intakes,and fecal and urine output, of individual animals. Rats had unrestrictedaccess to diets and drinking water throughout the study.

At the end of the treatment period rats were anaesthetized and varioussamples, including intestinal contents, collected and stored to awaitbiochemical analysis using standardized laboratory techniques.

The diets that were fed were based on the AIN-93G formulation and assuch comprised a uniform composition of macronutrients and fibre as NSP(at ˜15% w/w). A vitamin and mineral mixture added to ensure that alldiets were nutritionally complete. Four of the 5 diets containedoligosaccharide extracts from barley or chicory added at either 2% or 5%(by weight of diet).

Results:

Food intake was similar among treatment groups, although final liveweights were greater for all treatment groups compared to the control.Fecal output was greater for all treatment groups relative to controlsand related positively to level of dietary inclusion of the variousextracts. Treatment differences in caecal digesta weight mirrored thosefor faecal output. Consumption of treatment extracts was associated withacidification of caecal digesta. FIG. 3 shows caecal pools of individualand total SCFA. The total amount of SCFA was greater for the treatmentscompared to controls, and the responses to barley grain extract (at thecorresponding level of dietary inclusion) were comparable to those ofoligofructose. In summary, the barley grain mutant 292 extracts producedcomparable results to those of the corresponding oligofructosestandards.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 7 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1Hordeum vulgare subsp. vulgare starch synthase II mRNA, complete cDNAsequence. Accession No. AY133249, 2972 nucleotides, protein codingregion: nucleotides 114-2522, on chromosome 7 of barley. 2 amino acidsequence of starch synthase II encoded by SEQ ID NO: 1; 802 amino acids3 Triticum aestivum starch synthase IIa mRNA, complete cDNA sequence,Accession No. AF155217. 2842 nucleotides, protein coding region:nucleotides 89-2488 Reference: Li et al., Plant Physiol. 120: 1147-1156,1999. 4 amino acid sequence of starch synthase IIa encoded by SEQ ID NO:3: 799 amino acids 5 Triticum aestivum cDNA sequence for starch synthaseIIa-2 (wSSIIa-2 gene). Accession No. AJ269503. 2780 nucleotides, proteincoding region: nucleotides 55-2454; mature peptide: nucleotides 230-2451Reference: Gao and Chibbar, Genome 43: 768-775, 2000. 6 Triticumaestivum amino acid sequence encoded by wSSIIa gene for starch synthaseIIa (EC. 2.4.1.21), precursor 799 amino acids. 7 Triticum aestivumwSSIIa-B gene for starch synthase IIa, genomic sequence from B genome,Accession No. AB201446, exons: 246-506, 611-1316, 1407-1471, 2287-2364,2454-2564, 2662-2706, 3085-3258, 5855- 6811. Reference: Shimbata et al.,Theor. Appl. Genet. 111: 1072-1079, 2005. 8 amino acid sequence ofSSIIa-B encoded by SEQ ID NO: 7: 798 amino acids 9 Triticum aestivumwSSIIa-D gene for starch synthase IIa-D, predicted amino acid sequence,Accession No. AB201447: 799 amino acids 10 Sorghum bicolor starchsynthase IIa complete cDNA sequence, Accession No. EU620718, 2400nucleotides, protein coding region: 31-2262 11 amino acid sequenceencoded by nucleotides 31 to 2262 of SEQ ID NO: 10 12 Oryza sativa mRNAfor starch synthase IIa, complete cDNA sequence, Accession No. AB115918,2433 nucleotides, protein coding region: 1-2433. Reference: Nakamura etal., Plant Mol. Biol. 58: 213- 227, 2005. 13 amino acid sequence encodedby nucleotides 1 to 2432 of SEQ ID NO: 12: 810 amino acids 14 Zea maysZmSSIIa mRNA sequence, Accession No: BT023979, 2248 nucleotidesReference: Lai et al., Genome Res. 14: 1932-1937, 2004. 15 Oryza sativaSSIIb sequence of cDNA clone: Accession No. AK066446, 2645 nucleotides,Reference: Yamakawa et al., Plant Physiol 144: 258- 277, 2007. 16 Oryzasativa soluble starch synthase II-2 mRNA, complete cDNA sequence,Accession No. AF395537, 2394 nucleotides, protein coding region:nucleotides 34- 2118. 17 amino acid sequence encoded by nucleotide of34-2118 of SEQ ID NO: 16. Accession No. AAK81729, 694 amino acids 18Triticum aestivum starch synthase IIb precursor, cDNA sequence, 2025nucleotides Accession No. EU333947, protein coding region: 1-2025. 19amino acid sequence encoded by nucleotide 1 to 2025 of SEQ ID NO: 18:674 amino acids 20 Sorghum bicolor starch synthase IIb precursor, mRNA,complete cDNA sequence, Accession No. EU620719, 2302 nucleotides,protein coding region: 36-2150. 21 amino acid sequence encoded bynucleotide 1 to 2025 of SEQ ID NO: 20: 704 amino acids 22 Zea maysstarch synthase IIb- precursor, mRNA, complete cDNA sequence, AccessionNo. EF472249, protein coding region nucleotides 74-2188, 2569nucleotides 23 amino acid sequence encoded by nucleotide 1 to 2025 ofSEQ ID NO: 22: 704 amino acids

TABLE 8 Amino acid sub-classification Sub-classes Amino acids AcidicAspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic:Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine,Histidine Small Glycine, Serine, Alanine, Threonine, ProlinePolar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, TryptophanAromatic Tryptophan, Tyrosine, Phenylalanine Residues that influenceGlycine and Proline chain orientation

TABLE 9 Exemplary and Preferred Amino Acid Substitutions OriginalEXEMPLARY PREFERRED Residue SUBSTITUTIONS SUBSTITUTIONS Ala Val, Leu,Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu CysSer Ser Gln Asn, His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn,Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleu Leu Leu Norleu,Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe LeuPhe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp TyrTyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu

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The invention claimed is:
 1. A method of identifying a variety of wheatgrain which has increased levels of fructan comprising: (i) obtainingwheat grain of a mutant hexaploid wheat plant grown in a field, whereinsaid wheat grain has a reduced level or activity of an endogenous starchsynthase (SSII) polypeptide relative to grain from a wild-type wheatplant grown under the same environmental conditions as the mutanthexaploid wheat plant, wherein said wheat grain is homozygous for nullalleles of an SSII gene in each of the A, B, and D genomes, wherein thehomozygous alleles of the SSII gene of one or more of the A, B, and/or Dgenome(s) comprises a single base-pair substitution, insertion, ordeletion; (ii) determining the amount of fructan in the wheat grain; and(iii) if the wheat grain comprises at least 3% fructan as a percentageof the wheat grain weight, selecting the wheat grain.